U.S. patent number 11,085,932 [Application Number 15/766,489] was granted by the patent office on 2021-08-10 for periostin fragments and use thereof.
This patent grant is currently assigned to UNIVERSITE DE GENEVE. The grantee listed for this patent is UNIVERSITE DE GENEVE. Invention is credited to Nicolas Bonnet, Serge Ferrari, Daniel S. Spellman.
United States Patent |
11,085,932 |
Ferrari , et al. |
August 10, 2021 |
Periostin fragments and use thereof
Abstract
The present invention relates to biological markers of metabolic
bone diseases or disorders such as osteoporosis and related methods
using thereof. The invention further relates to the use of those
biomarkers and related material and assays in the diagnosis of
subjects at risk of developing such disorders or monitoring the
effects of a treatment thereof.
Inventors: |
Ferrari; Serge (Saconnex
d'Arve/Geneve, CH), Bonnet; Nicolas (Cranves-Sales,
FR), Spellman; Daniel S. (West Point, PA) |
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE DE GENEVE |
Geneva |
N/A |
CH |
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Assignee: |
UNIVERSITE DE GENEVE (Geneva,
CH)
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Family
ID: |
57121255 |
Appl.
No.: |
15/766,489 |
Filed: |
October 7, 2016 |
PCT
Filed: |
October 07, 2016 |
PCT No.: |
PCT/EP2016/074086 |
371(c)(1),(2),(4) Date: |
April 06, 2018 |
PCT
Pub. No.: |
WO2017/060482 |
PCT
Pub. Date: |
April 13, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180284135 A1 |
Oct 4, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62238901 |
Oct 8, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
14/475 (20130101); C07K 16/18 (20130101); G01N
33/6893 (20130101); G01N 2800/52 (20130101); G01N
2800/50 (20130101); G01N 2800/108 (20130101) |
Current International
Class: |
G01N
33/68 (20060101); C07K 16/18 (20060101); C07K
14/475 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2008/021290 |
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Feb 2008 |
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WO |
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Other References
Helali, A. M. et al. "Cathepsin K Inhibitors: A Novel Target but
Promising Approach in the Treatment of Osteoporosis" Current Drug
Targets, 2013, pp. 1591-1600, vol. 14, No. 13. cited by applicant
.
Gineyts, E. et al. "The C-Terminal Intact Forms of Periostin (iPTN)
Are Surrogate Markers for Osteolytic Lesions in Experimental Breast
Cancer Bone Metastasis" Calcified Tissue International, Jun. 18,
2018, pp. 1-14. cited by applicant .
Bonnet, N. "Increased Periostin in Cortical Bone of Cathepsin K
Knock-Out Mice is Responsible for Higher Cortical Bone Mass and
Strength" ASBMR Annual Meeting 2014, Poster Sessions, Presentation
No. SU0172, Abstract Only, pp. 1-2. cited by applicant .
Bonnet, N. et al. "Increased periostin in cortical bone of
cathepsin K knock out mice is responsible for higher cortical bone
mass and strength" ASBMR Annual Meeting 2014, Presentation No.
SU0172, Poster, p. 1. cited by applicant .
Anastasilakis, A.D. et al. "Circulating Periostin Levels do not
Diff er Between Postmenopausal Women with Normal and Low Bone Mass
and are not Affected by Zoledronic Acid Treatment" Home Metab Res,
2014, pp. 145-149, vol. 46. cited by applicant .
Bonnet, N. et al. "The Matricellular Protein Periostin Is Required
for Sost, Inhibition and the Anabolic Response to Mechanical
Loading and Physical Activity" The Journal of Biological Chemistry,
Dec. 18, 2009, pp. 35939-35950, vol. 284, No. 51. cited by
applicant .
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Endocrinology, 2016, pp. 75-82, vol. 432. cited by applicant .
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Degrading Periostin" Journal of Bone and Mineral Research, 2017,
pp. 1-10. cited by applicant .
Bonnet, N. et al. "Serum Levels of a Cathepsin-K Generated
Periostin Fragment Predict Incident Low-Trauma Fractures in
Postmenopausal Women Independently of BMD and FRAX" Journal of Bone
and Mineral Research, 2017, pp. 1-7. cited by applicant .
Chapurlat, R. D. et al. "Novel biological markers of bone: from
bone metabolism to bone physiology" Rheumatology, 2016, pp.
1714-1725, vol. 55. cited by applicant .
Chevalley, T. et al. "Fracture history of healthy premenopausal
women is associated with a reduction of cortical microstructural
components at the distal radius" Bone, 2013, pp. 377-383, vol. 55.
cited by applicant .
Contie, S. et al. "Development of a New ELISA for Serum Periostin:
Evaluation of Growth-Related Changes and Bisphosphonate Treatment
in Mice" Calcif Tissue Int, Jun. 22, 2010, pp. 1-10. cited by
applicant .
Dodds, R. A. et al. "Human Osteoclast Cathepsin K Is Processed
Intracellularly Prior to Attachment and Bone Resorption" Journal of
Bone and Mineral Research, 2001, pp. 478-486, vol. 16, No. 3. cited
by applicant .
Duong, L.T. et al. "Cathepsin K Inhibition: A New Mechanism for the
Treatment of Osteoporosis" Calcif Tissue Int, 2016, pp. 381-397,
vol. 98. cited by applicant .
Durosier, C. et al. "Association of Circulating Sclerostin With
Bone Mineral Mass, Microstructure, and Turnover Biochemical Markers
in Healthy Elderly Men and Women" Journal of Clinical Endocrinology
Metabolism, Sep. 2013, pp. 3873-3883, vol. 98, No. 9. cited by
applicant .
Garnero, P. "New Developments in biological markers of bone
metabolism in osteoporosis" Bone, 2014, pp. 46-55, vol. 66. cited
by applicant .
Garnero, P. et al. "Development of a New Immunoassay for Human
Cathepsin K-Generated Periostin Fragments as a Serum Biomarker for
Cortical Bone" Calcif Tissue Int, 2017, pp. 501-509, vol. 101.
cited by applicant .
Horiuchi, K. et al. "Identification and Characterization of a Novel
Protein, Periostin, with Restricted Expression to Periosteum and
Periodontal Ligament and Increased Expression by Transforming
Growth Factor .beta." Journal of Bone and Mineral Research, 1999,
pp. 1239-1249, vol. 14, No. 7. cited by applicant .
Lippuner, K. et al. "FRAX.RTM. assessment of osteoporotic fracture
probability in Switzerland" Osteoporos Int, 2010, pp. 381-389, vol.
21. cited by applicant .
Meier, C. et al. "Serum Cathepsin K Concentrations Reflect
Osteoclastic Activity in Women with Postmenopausal Osteoporosis and
Patients with Paget's Disease" Clin Lab, 2006, pp. 1-10, vol. 52.
cited by applicant .
Merle, B. et al. "The multiple facets of periostin in bone
metabolism" Osteoporos Int, 2012, pp. 1199-1212, vol. 23. cited by
applicant .
Shengyu, L. et al. "Histochemical examination of cathepsin K, MMP1
and MMP2 in compressed periodontal ligament during orthodontic
tooth movement in periostin deficient mice" J Mol Hist, 2014, pp.
303-309, vol. 45. cited by applicant .
Sun, S. et al. "The development and characterization of an ELISA
specifically detecting the active form of cathepsin K" Clinical
Biochemistry, 2013, pp. 1601-1606, vol. 46. cited by applicant
.
Written Opinion in International Application No. PCT/EP2016/074086,
dated Jan. 10, 2017, pp. 1-7. cited by applicant.
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Primary Examiner: Giere; Rebecca M
Attorney, Agent or Firm: Saliwanchik, Lloyd &
Eisenschenk
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage application of
International Patent Application No. PCT/EP2016/074086, filed Oct.
7, 2016, which claims the benefit of U.S. Provisional Patent
Application No. 62/238,901, filed Oct. 8, 2015.
The Sequence Listing for this application is labeled "Seq-List.txt"
which was created on Mar. 23, 2018 and is 15 KB. The entire content
of the sequence listing is incorporated herein by reference in its
entirety.
Claims
The invention claimed is:
1. An isolated periostin (POSTN) fragment consisting of SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or variants thereof, wherein
said variant has one or more amino acid that is deleted or is a
conservative substitution.
2. The POSTN fragment according to claim 1, said POSTN fragment
consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, or a variant thereof.
3. The POSTN fragment according to claim 1, said POSTN fragment
consisting of SEQ ID NO: 2.
4. A method for detecting a POSTN fragment from a biological fluid
sample of a mammalian subject comprising the steps of: (a)
providing a biological fluid sample obtained from a mammalian
subject; (b) providing a solid support having bound thereto at
least one POSTN fragments according to claim 1; (c) bringing said
solid support into contact with said biological fluid sample; (d)
bringing said solid support into contact with at least one antibody
specific for said POSTN fragment, wherein the contacting is under
conditions sufficient for binding of said POSTN fragment present in
said biological fluid sample to said at least one antibody through
antigen-antibody interactions; (e) washing the solid matrix for
removing any unbound antibody from the surface of the said solid
matrix; and (f) detecting the presence of an antigen-antibody
complex bound to the said solid matrix, wherein the presence of the
said complex is indicative that the biological fluid sample
contains one or more POSTN fragment.
5. A method for detecting a POSTN fragment in a biological fluid
sample of a mammalian subject comprising the steps of: (a)
providing a biological fluid sample obtained from a mammalian
subject; (b) bringing said biological fluid sample into contact
with a solid matrix where at least one antibody is bound to,
wherein said at least one antibody is specific for a POSTN fragment
according to claim 1, and wherein the contacting is under
conditions sufficient for binding a POSTN fragment present in the
said biological fluid sample to said at least one antibody through
antigen-antibody interactions; (c) removing the biological fluid
sample from the solid matrix for removing any unbound material from
the surface of the said solid matrix; and (d) detecting the
presence of an antigen-antibody complex bound to said solid matrix,
wherein the presence of the said complex is indicative that the
biological fluid sample contains one or more POSTN fragment(s).
6. The method according to claim 4, wherein the said at least one
antibody is specific for a POSTN fragment consisting of SEQ ID NO:
2.
7. The method according to claim 4, wherein a combination of
antibodies or of variants thereof is bound to the said solid matrix
and where the combination comprises: a) one antibody specific for a
POSTN fragment consisting of SEQ ID NO: 2; and b) at least one
antibody specific for a POSTN fragment consisting of SEQ ID NO: 3,
SEQ ID NO: 4, or SEQ ID NO: 5, or a variant thereof, wherein said
variant has one or more amino acid that is deleted or is a
conservative substitution.
8. The method according to claim 4, wherein the said at least one
antibody is specific for a POSTN fragment of 6 to 20 amino acids
that includes SEQ ID NO: 2.
9. The method according to claim 4, wherein the presence of one or
more POSTN fragment(s), is indicative of a metabolic bone disorder
or a risk of fracture.
10. An isolated nucleic acid molecule encoding a POSTN fragment
according to claim 1.
11. An isolated antibody or fragment thereof specific for a POSTN
fragment or variant thereof according to claim 1.
12. An isolated nucleic acid molecule encoding an antibody or
fragment thereof according to claim 11.
13. A recombinant expression vector comprising a nucleic acid
molecule according to claim 12.
14. A host cell comprising a recombinant expression vector
according to claim 13.
15. A process for producing antibodies or fragments thereof
comprising culturing a host cell transformed with an expression
vector comprising a nucleic acid sequence that encodes the
antibodies or fragments of claim 11 under conditions sufficient to
promote expression of said antibodies or fragments thereof.
16. A kit for detecting a POSTN fragment in a biological fluid
sample, the kit comprising at least one POSTN fragment according to
claim 1.
17. The kit according to claim 16, said kit comprising: a) a solid
support having bound thereto at least one POSTN fragment or a
variant thereof, wherein said variant has one or more amino acid
that is deleted or is a conservative substitution; and optionally:
b) at least one antibody specific to said POSTN fragment or a
variant thereof, wherein said variant has one or more amino acid
that is deleted or is a conservative substitution; c) at least one
unbound POSTN fragment or a variant thereof, wherein said variant
has one or more amino acid that is deleted or is a conservative
substitution; and/or d) at least one detection agent for detecting
the complex that forms between said at least one POSTN fragment and
an antibody binding said at least one POSTN fragment of a variant
thereof, wherein said variant has one or more amino acid that is
deleted or is a conservative substitution.
18. The kit according to claim 17, wherein said kit is an ELISA kit
or a competitive ELISA kit.
19. An immunoassay preparation comprising at least one antibody
according to claim 11 for the detection of a POSTN fragment in a
biological fluid sample.
20. A method of monitoring the effect of a treatment of a metabolic
bone disorder in a subject comprising the steps of a method
according to claim 4.
21. The method according to claim 20, wherein a metabolic bone
disorder is osteoporosis.
22. A method of making a periostin (POSTN) peptide fragment
comprising incubating human recombinant POSTN with Cathepsin K
(CatK) and generating POSTN peptide fragments.
23. The method according to claim 22, wherein POSTN and CatK are
incubated in a POSTN to CatK ratio from about 100:1 to about
22:1.
24. The method according to claim 22, wherein POSTN and CatK are
incubated at a temperature of about 35.degree. to about 38.degree.
C.
25. The method according to claim 22, wherein said CatK comprises
SEQ ID NO: 19.
Description
FIELD OF THE INVENTION
The present invention relates generally to the field of biological
markers and in particular biological markers of metabolic bone
diseases such as osteoporosis. In particular, the invention relates
to methods useful for the treatment and diagnosis of patients at
risk of osteoporosis and monitoring progression of treatment
thereof.
BACKGROUND OF THE INVENTION
The periosteum covers long bones and plays an important role in
controlling the bone diameter and bone strength, however
non-invasive methods of assessment of periosteal metabolism are not
yet developed (Contie et al., 2010, Calcif. Tissue Int., 87(4):
341-50; Garnero, 2014, Bone, 66: 46-55). Periostin (POSTN) is a
90-KDa matricellular gamma-carboxyglutamic acid protein
preferentially expressed in periosteum and bone mesenchymal stem
cells but also in collagen rich tissues subjected to mechanical
strain, such as periodontal ligaments, heart valves, tendons and
skin (Contie et al., 2010, supra; Merle et al., 2012, Osteoporos.
Int., 23: 1199-212). Periostin, initially known also as
osteoblast-specific factor-2, is an 811-amino acid protein
comprising an N-terminal secretory signal peptide, followed by an
Emilin (EMI) cysteine rich domain, four internal homologous repeats
so called Fasciclin-1 (FAS-1) domains, and a C-terminal hydrophilic
and variable domain (Merle et al., 2012, supra). Periostin is an
important regulator of osteoblastic activity, bone formation and
regulation of bone strength by regulating collagen crosslinking,
particularly at cortical sites and also osteoclastic activity by
increasing Osteoprotegerin but it is not specific to bone tissue.
POSTN exists in different forms due to alternative splicing (7
isoforms), gamma-carboxylation and dimerization, currently it is
not known if one of the isoforms could be specific only for bone
tissue (Merle et al., 2012, supra). Some data on the function of
POSTN in bone metabolism have been obtained from the examination of
POSTN deficient mice (Bonnet et al., 2009, J. Biol. Chem., 284:
35939-50). These mice develop periodontis and osteoporosis with
lower bone mineral density (BMD), altered microarchitecture and
decreased bone strength. This study has also shown that POSTN is an
important mediator of the effects of mechanical factors and
parathyroid hormone on cortical BMD and bone strength by modulating
the canonical wnt signaling pathway with a down regulation of
sclerostin expression (Bonnet et al., 2009, supra). POSTN is
secreted by osteocytes, osteoblasts and to low extend by
osteoclasts and has been proposed to be a potential bone marker of
cortical structure parameters (Garnero, 2014, supra). Because POSTN
is a secreted protein it can be detected e.g. in peripheral blood
and thus POSTN immunoassays have been developed for rodents (Contie
et al., 2010, supra) and humans (Anastasilakis et al., 2014, Horm.
Metab. Res., 46: 145-9). However, these assays detect serum POSTN
levels independent of their origin or isoform type and thus are
non-specific. Various antibodies have been developed targeting
different POSTN epitopes as represented on FIG. 1: polyclonal
antibodies to human POSTN (Uscn Life Science, Inc.; Product No.:
SEH339Hu) target the region of amino acids 97-230 (FAS 1-1) and
monoclonal antibodies to human and mouse POSTN (Adipogen AG.;
Product No.: BI-20433) target the region of amino acids 234-365
(FAS 1-2). However, those antibodies do not recognize a specific
isoform of POSTN nor a tissue specific fragment. Among identified
biochemical markers of bone metabolism, CTX (cross-linked
C-terminal telopeptide of type I collagen) is used as a marker of
bone resorption and P1NP (procollagen type 1 N-terminal propeptide)
is used as a marker of bone remodelling. However, those markers are
not specific of the trabecular/cortical compartments. Therefore,
there is a need to identify markers and develop assays directed to
assessment of metabolism of cortical bone that are more specific
than detection of overall serum POSTN levels. Further, there are
currently no anabolic agents able to increase trabecular bone
formation except parathyroid hormone (PTH) which is known for
having severe side effects. Therefore, there is a need to develop
tools for the monitoring of the trabecular/cortical compartments in
view of the identification of alternative anabolic agents.
Cathepsin K (CatK) is an enzyme, specifically a protease, which is
defined by its high specificity for kinins and is involved in bone
metabolism. It is a lysosomal enzyme synthesized as a procathepsin
K, which is activated in the lysosomes through a process involving
autocatalytic cleavage at low pH, and then released into the
resorption lacunae (Dodds et al., 2001, J. Bone Miner. Res., 16:
478-86). Cathepsin K is one of the main catalytic enzymes expressed
and secreted by the osteoclasts. It is critical in
osteoclast-mediated bone resorption as it plays a predominant role
in the degradation of bone type I collagen (Garnero, 2014, supra).
Consequently CatK metabolic pathway became a drug target in
treatment of osteoporosis, e.g. CatK inhibitors, such as odanacatib
have been developed (Helali et al., 2013, Curr. Drug Targets, 14:
1591-600).
Independently, some of CatK can be further released in the
circulation and thus it was speculated to be a biological marker of
osteoclast activity. Several assays for detection of CatK from
blood have been developed, although it remains unclear whether they
detect the proenzyme, the active form or both (Garnero, 2014,
supra). By using such tests, it has been shown that serum CatK is
increased in patients with osteoporosis (Meier et al., 2006, Clin.
Lab, 52: 1-10). Further, by using an assay directed to detecting
only the active form of CatK, no change in serum of active
cathepsin K levels could be observed in postmenopausal women
treated with anti-resorptive therapy (Sun et al., 2013, Clin.
Biochem., 46: 1606-6). Cathepsin K inhibitors are currently
developed for the treatment of osteoporosis and other skeletal
disorders associated with excessive bone remodeling (Odanacatib,
Merck & Co., Inc.) but the currently available biomarkers do
not offer an appropriate tool to monitor the effects of those
inhibitors on the remodeling activity in the trabecular/cortical
compartments.
Therefore, it is currently of interest to develop assays that
measure specific markers of cathepsin K activity in relation to
bone cortical structure in order to monitor pathophysiological
metabolism of bone in patients with bone disorders or at risk of
developing those and/or to monitor the effect of treatments of
metabolic bone disorders.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected finding that POSTN
is a substrate for CatK-mediated degradation that is restricted to
bones, specifically to bone cortical region. CatK-dependent
cleavage products (POSTN fragments) could therefore serve as
biomarkers of bone metabolic changes related to bone disorders such
as osteoporosis.
In particular, it has been identified POSTN fragments which are
specific of the bone tissue as opposed to intact POSTN. More
particularly, the invention is based on the finding of markers of
the bone quality, in particular in term of the cortical
microstructure and bone matrix, which markers can be used as
reliable predictive indicators of the risk of fracture in a
subject. The present invention is directed towards a method of
detection of POSTN fragments from a biological fluid sample of a
mammalian subject and related antibodies, assay and kits suitable
for the diagnosis of patients at risk of developing a metabolic
bone disorder such as osteoporosis and monitoring the effect of a
treatment thereof.
One aspect of the invention provides an isolated peptide POSTN
fragment or variants thereof.
Another aspect of the invention relates to an isolated peptide
POSTN fragment or variants thereof, or formulation thereof
according to the invention for use in the prevention and/or
treatment of a metabolic bone disorder such as osteoporosis.
Another aspect of the invention provides a process of producing
POSTN fragments or variants thereof comprising incubating POSTN
with CatK.
Other aspect of the invention relates to an isolated nucleic acid
molecule encoding a POSTN fragment or variant thereof as described
herewith.
Another aspect of the invention relates to an isolated antibody or
fragment thereof specific for POSTN fragment or variant
thereof.
Another aspect of the invention relates to an isolated nucleic acid
molecule encoding an antibody or fragment thereof as described
herewith.
Another aspect of the invention relates to a recombinant expression
vector comprising said nucleic acid molecule, and to a host cell
comprising said recombinant vector, respectively.
Another aspect of the invention relates to a process for producing
antibodies or fragments thereof as described herewith comprising
culturing a host cell transformed with an expression vector
comprising a nucleic acid sequence that encodes said antibodies or
fragments thereof under conditions sufficient to promote expression
of said antibodies or fragments thereof.
Next aspect of the invention provides a method for detecting a
POSTN fragment from a biological fluid sample of a mammalian
subject comprising the steps of: (a) providing a biological fluid
sample obtained from a mammalian subject; (b) bringing said
biological fluid sample into contact with a solid matrix where at
least one antibody is bound to, wherein the contacting is under
conditions sufficient for binding a POSTN fragment of the invention
present in said biological fluid sample to said at least one
antibody through antigen-antibody interactions and wherein said at
least one antibody is specific for POSTN fragment of the invention
or any variant thereof; (c) removing the biological fluid sample
from the solid matrix for removing any unbound POSTN fragment of
the invention from the surface of the said solid matrix; (d)
detecting the presence of an antigen-antibody complex bound to the
said solid matrix, wherein the presence of the said complex is
indicative that the biological fluid sample contains one or more
POSTN fragments of the invention.
Next aspect of the invention provides a method for detecting a
POSTN fragment from a biological fluid sample of a mammalian
subject comprising the steps of: (a) providing a biological fluid
sample obtained from a mammalian subject; (b) bringing said
biological fluid sample into contact with at least one antibody,
wherein the contacting is under conditions sufficient for binding a
POSTN fragment of the invention present in said biological fluid
sample to said at least one antibody through antigen-antibody
interactions and wherein said at least one antibody is specific for
POSTN fragment of the invention or any variant thereof; (c)
bringing sample obtained under step b) into contact with a solid
matrix where at least one POSTN fragment of the invention is bound
to, wherein the contacting is under conditions sufficient for
binding an antibody specific for POSTN fragment of the invention
present in said sample to said at least one POSTN fragment of the
invention through antigen-antibody interactions; (d) washing the
solid matrix for removing any unbound antibody from the surface of
the said solid matrix; (e) detecting the presence of an
antigen-antibody complex bound to the said solid matrix, wherein
the presence of the said complex is indicative that the biological
fluid sample contains one or more POSTN fragments of the
invention.
In another aspect, the invention provides a method for detecting a
POSTN fragment from a biological fluid sample of a mammalian
subject comprising the steps of: (a) providing a biological fluid
sample obtained from a mammalian subject; (b) providing a solid
support having bound thereto at least one POSTN fragments of the
invention; (c) bringing said solid support into contact with said
biological fluid sample; (d) bringing said solid support into
contact with at least one antibody specific for POSTN fragment of
the invention or any variant thereof, wherein the contacting is
under conditions sufficient for binding a POSTN fragment of the
invention present in said biological fluid sample to said at least
one antibody through antigen-antibody interactions; (e) washing the
solid matrix for removing any unbound antibody from the surface of
the said solid matrix; (f) detecting the presence of an
antigen-antibody complex bound to the said solid matrix, wherein
the presence of the said complex is indicative that the biological
fluid sample contains one or more POSTN fragments of the
invention.
In another aspect, the invention provides an immunoassay
preparation for the detection of a POSTN fragment of the invention
comprising at least one antibody according to the invention.
Another aspect of the invention relates a kit for detecting a POSTN
fragment of the invention in a biological fluid sample, the kit
comprising at least one antibody according to the invention or a
variant thereof.
Another aspect of the invention provides a method of monitoring the
effects of a treatment of metabolic bone disorders, comprising
detection of a POSTN fragment of the invention in a biological
fluid sample from a subject under treatment.
Another aspect of the invention provides a method of diagnosis of
subjects at risk of developing a metabolic bone disorder such as
osteoporosis comprising detection of a POSTN fragment of the
invention.
Another aspect of the invention provides an indirect method of
evaluating cortical bone volume and porosity comprising detection
of a POSTN fragment of the invention.
DESCRIPTION OF THE FIGURES
FIG. 1 is schematic representation of the amino acid sequence of
POSTN with its different domains: the EMI domain, the four FAS
domains and the C terminal (C-Term) variable region. Numbers
indicate amino acid positions on the mature POSTN amino acid
sequence. The peptide signal at N terminal is indicated as cleaved.
Ab1: commercial antibodies to human POSTN (USCN) targeting 97-230
(FAS 1-1), Ab2: commercial antibodies to human and mouse POSTN
(Adipogen AG) targeting 234-365 (FAS 1-2).
FIG. 2 shows SDS-PAGE gel of CatK digested human POSTN (A-B). A:
Digestion lasting for 1 h, 2 h, 3 h or overnight with constant
POSTN/CatK ratio of 50:1 as described in Example 1. Molecular
weight (MW) in kDa of peptides is indicated. B: Digestion of
constant amount of POSTN for 4 h, at 37.degree., with increasing
amount of CatK; POSTN/CatK ratios ranging from 1000:1 to 11:1 are
indicated. A-B: POSTN fragments were visualized by silver staining;
dominant band at around 90 kDa corresponds to POSTN.
FIG. 3 shows twenty-one different POSTN fragments separated in a
single LC-MS/MS analysis as described in Example 2. A: The
sequences of the identified POSTN fragments are indicated on the
corresponding signal. The relative % absorbance is indicated. B:
LC-MS/MS measurement of the two major CatK-cleavage POSTN fragments
obtained with POSTN/CatK ratio ranging from 250:1 to 5:1. 1-
Peptide 1 (SEQ ID NO: 2), 2- Peptide 2 (SEQ ID NO: 3), 3- Peptide 3
(SEQ ID NO: 4), 4- Peptide 4 (SEQ ID NO: 5), 5- Peptide 5 (SEQ ID
NO: 6), 6- Peptide 6 (SEQ ID NO: 7), 7- Peptide 7 (SEQ ID NO: 8),
8- Peptide 8 (SEQ ID NO: 9), 9- Peptide 9 (SEQ ID NO: 10), 10-
Peptide 10 (SEQ ID NO: 11), 11- Peptide 11 (SEQ ID NO: 12), 12-
Peptide 12 (SEQ ID NO: 13), 13- Peptide 13 (SEQ ID NO: 14), 14-
Peptide 14 (SEQ ID NO: 15), 15- Peptide 15 (SEQ ID NO: 16), 16-
Peptide 16 (SEQ ID NO: 17), 17- Peptide 17 (SEQ ID NO: 18), 18-
Peptide 18 (SEQ ID NO: 22), 19- Peptide 19 (SEQ ID NO: 23), 20-
Peptide 20 (SEQ ID NO: 24), 21- Peptide 21 (SEQ ID NO: 25).
FIG. 4 shows the time course of POSTN fragments appearance between
15 min to 2 h during CatK-dependent POSTN digestion as described in
Example 3, at POSTN/CatK ratio of 50:1. A: the dotted area is shown
on panel B. POSTN fragments indicated as in FIG. 3.
FIG. 5 shows an overview of CatK-cleavage POSTN fragments sites in
human POSTN (A) (SEQ ID NO: 1) and their abundance as measured by
LC-MS/MS (B) (SEQ ID NOs: 2, 6, 3, 5, 4, 7-11, 13, 12 and 14-18,
respectively).
FIG. 6 shows immunostaining of osteocytes and lacuno canalicular
system from mouse bone detected in presence of anti-SEQ ID NO: 2 as
described in Example 6. A: control; B: in presence of anti-SEQ ID
NO: 2 antibodies at 1/200000; C: in presence of anti-SEQ ID NO: 2
antibodies at 1/10000.
FIG. 7 shows ELISA standard curve (OD at 450 nm versus peptide 1
concentration [c] in ng/ml) as described in Example 7.
FIG. 8 shows ELISA test read out (OD at 450 nm versus peptide
concentration [c] in ng/ml) described in Example 7 for peptide 1
(rhombus), control peptide of SEQ ID NO: 20 (square), control
peptide of SEQ ID NO: 21 (triangle) and POSTN (SEQ ID NO: 1,
cross).
FIG. 9 represents serum levels of peptide 1 [c.sub.i] versus sPOSTN
serum levels [c.sub.2] (ng/ml) measured in Example 8.
FIG. 10 shows tibia Ct. area and stiffness distribution of low,
middle and high tertile of sPOSTN (A, B) and Peptide 1 to sPOSTN
ratio (C, D) as described in Example 9.
FIG. 11 represents serum levels of peptide 1 [c.sub.1] versus
sPOSTN serum levels [c.sub.2] in ng/ml measured with commercial
kits 1 (A, n=324) or 2 (B, n=176) as described in Example 11.
FIG. 12 shows ROC curves comparison for A: BMD femoral neck
(AUC=0.555; grey) versus BMD femoral neck and peptide 1 (AUC=0.692;
black) p=0.005; B: FRAX (AUC=0.667; grey) versus FRAX and peptide 1
(AUC=0.730; black) as described in Example 12.
DETAILED DESCRIPTION
"Polypeptide" is understood as a peptide, an oligopeptide, an
oligomer or a protein comprising at least two amino acids joined to
each other by a normal or modified peptide bond, such as in the
cases of the isosteric peptides, for example. A polypeptide can be
composed of amino acids other than the 20 amino acids defined by
the genetic code. A polypeptide can equally be composed of amino
acids modified by natural processes, such as post-translational
maturation processes or by chemical processes, which are well known
to a person skilled in the art. Such modifications are fully
detailed in the literature. These modifications can appear anywhere
in the polypeptide: in the peptide skeleton, in the lateral chain
or even at the carboxy- or amino-terminal ends. For example,
polypeptide modifications are understood to include
phosphorylations, glycolysation, isomerisation, gamma
carboxylation, dimerisation. Such modifications are fully detailed
in the literature (Proteins Structure and Molecular Properties
(1993) 2.sup.nd Ed., T. E. Creighton, New York; Post-translational
Covalent Modifications of Proteins (1983) B. C. Johnson, Ed.,
Academic Press, New York; Seifter et al. (1990) Analysis for
protein modifications and nonprotein cofactors, Meth. Enzymol. 182:
626-646 and Rattan et al., (1992) Protein Synthesis:
Post-translational Modifications and Aging, Ann NY Acad Sci, 663:
48-62).
The term "periostin", abbreviated "POSTN", also known as
osteoblast-specific factor-2, is a matricellular
gamma-carboxyglutamic acid polypeptide protein preferentially
expressed in periosteum and bone mesenchymal stem cells. POSTN can
also be expressed in collagen rich tissues subjected to mechanical
strain such as periodontal ligaments, heart valves and tendons.
POSTN is an 811-amino acid protein of SEQ ID NO: 1, comprising an
N-terminal secretory signal peptide, followed by an Emilin (EMI)
cysteine rich domain, four internal homologous repeats so called
Fasciclin-1 (FAS-1) domains, and a C-terminal hydrophilic domain
and variable domain. The recombinant human POSTN (Horiuchi et al.,
1999, J Bone Miner Res 14:1239) comprises an N-terminal secretory
signal peptide (residues 1-21 from SEQ ID NO: 1) that is not
present in mature form of POSTN, EMI domain (residues 40-94 from
SEQ ID NO: 1), 4 of FAS-1 domains (respectively residues 97-230,
234-365, 368-492 and 496-628 from SEQ ID NO: 1) and C-terminal
variable domain (residues 628-836 from SEQ ID NO: 1), FIG. 1. The
term "periostin fragment" or "POSTN fragment" refers to a
polypeptide POSTN fragment according to the invention obtained
after CatK-dependent degradation in bone.
The term "cathepsin K", abbreviated "CatK", as referred to herein,
means an enzyme of reference EC:3.4.22.38, specifically a protease,
which is defined by its high specificity for kinins and is involved
in bone resorption. CatK catabolizes elastin, collagen, and gelatin
and allows it to break down bone and cartilage. CatK is highly and
selectively expressed by osteoclast and is critical in
osteoclast-mediated bone resorption. An example sequence of human
CatK is provided as SEQ ID NO: 19.
The term "isolated" is used to indicate that the molecule is free
of association with other proteins or polypeptides, for example as
a purification product. Further, "isolated peptide" or "isolated
polypeptide" is understood as a polypeptide such as POSTN fragment
which is isolated from the human body or otherwise produced by a
technical process.
The term "variant" as referred to herein, means a polypeptide
substantially homologous to the original peptide sequence, but
which has at least one an amino acid sequence different from that
of the original sequence because of one or more deletions,
insertions or substitutions. Substantially homologous means a
variant amino acid sequence that is at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%
or at least 99% identical to the original amino acid sequences, as
disclosed above. The percentage identity of two amino acid
sequences can be determined by visual inspection and/or
mathematical calculation, or more easily by comparing sequence
information using known computer program used for sequence
comparison such as Clustal package version 1.83. A variant may
comprise a sequence having at least one conservatively substituted
amino acid, meaning that a given amino acid residue is replaced by
a residue having similar physiochemical characteristics. Generally,
substitutions for one or more amino acids present in the original
polypeptide should be made conservatively. Examples of conservative
substitutions include substitution of one aliphatic residue for
another, such as Ile, Val, Leu, or Ala for one another, or
substitutions of one polar residue for another, such as between Lys
and Arg; Glu and Asp; or Gln and Asn. Other such conservative
substitutions, for example, substitutions of entire regions having
similar hydrophobicity characteristics, are well known (Kyte, et
al, 1982, J. Mol. Biol., 157: 105-131). For example, a
"conservative amino acid substitution" may involve a substitution
of a native amino acid residue with a non-native residue such that
there is little or no effect on the polarity or charge of the amino
acid residue at that position. Desired amino acid substitutions
(whether conservative or non-conservative) can be determined by
those skilled in the art at the time such substitutions are
desired. Exemplary amino acid substitutions are presented in Table
1 below:
TABLE-US-00001 TABLE 1 Original residues Examples of substitutions
Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln Asp (D) Glu
Cys (C) Ser, Ala GIn (Q) Asn Glu (E) Asp Gly (G) Pro, Ala His (H)
Asn, GIn, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu
(L) Ile, Val, Met, Ala, Phe, Norleucine Lys (K) Arg, GIn, Asn Met
(M) Leu, Ile, Phe Phe (F) Leu, Val, Ile, Ala, Tyr Pro (P) Ala, Gly
Ser (S) Thr, Ala, Cys Thr (T) Ser Tyr (Y) Trp, Phe, Thr, Ser Val
(V) Ile Met, Leu, Phe, Ala, Norleucine
The expression "biological fluid sample" refers to a clinical fluid
sample for testing which is taken from a body fluid from a mammal
such as saliva, bone marrow, blood, pleural liquid, sweat and
urine. For example, a biological fluid sample is a serum/plasma
sample from a human subject.
The expression "control sample" refers to a positive control or a
negative control sample. A negative control sample includes a body
fluid sample taken from a subject that is the same or homologous
species as the subject to be assayed for POSTN fragments and is
known to have normal biological state, e. g. without detectable
POSTN fragments or a solution which does not contain POSTN
fragments that are immunoreactive with at least one antibody
according to the invention. A negative control sample includes a
sample taken from a control subject. A positive control sample
includes a body fluid sample taken from a subject that is the same
or homologous species as the subject to be assayed for POSTN
fragments and is known to have detectable POSTN fragments according
to the invention or a solution which does contain POSTN fragments
that are immunoreactive with at least one antibody according to the
invention.
The term "antibody" as referred to herein designates a polypeptide
that binds to an antigen. This includes whole antibodies and any
antigen binding fragments. The term "antibody" is used in its
broadest sense and includes monoclonal antibodies, polyclonal
antibodies, human antibodies, humanized antibodies, chimeric
antibodies, and further engineered antibodies as long as the
characteristic properties of the invention are retained, in
particular the ability of binding to the target antigen, more
specifically to the epitope of POSTN fragment as the one recognized
by the antibodies of the invention. Examples of antibodies and
fragments thereof include a variable domain fragment ("Fv",
consisting of the VH and VL domains of a single arm of an
antibody), Fab fragment (monovalent fragment consisting of the VH,
VL, CH1 and CL domains), Fab.sub.2 fragment (bivalent), Fab.sub.3
fragment (trivalent), Fab' fragment (Fab with hinge region),
F(ab').sub.2 fragment (bivalent fragment including two Fab
fragments linked by a disulfide bridge at the hinge region), Fd
fragment (consisting of the VH and CH1 domains), rIgG (reduced IgG
or half-IgG), diabodies, triabodies, tetrabodies, minibodies,
monovalent antibodies, divalent or multivalent antibodies
comprising a fragment of more than one antibody, single chain
variable fragment (ScFv), bis-scFv (bispecific), and derivatives of
antibodies such as disulfide stabilized Fv fragments,
CDR-comprising peptides, as well as epitope-binding fragments of
any of the above (Holliger and Hudson, 2005, Nature Biotechnology,
23(9): 1126-1136). An antibody refers to a glycoprotein comprising
at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds, or an antigen-binding fragment
thereof. Each heavy chain comprises a heavy chain variable region
(VH) and a heavy chain constant region (CH). Each light chain
comprises a light chain variable region (VL) and a light chain
constant region (CL). In mammalians, the heavy chain can either be
alpha (.alpha.), delta (.delta.), epsilon (.epsilon.), gamma
(.gamma.) or mu (.mu.), which defines the class of antibody IgA,
IgD, IgE, IgG and IgM, respectively. In mammalians, the light chain
can either be lambda (.lamda.) or kappa (.kappa.). In mammalians,
depending on the class of antibody, the heavy chain constant region
comprises three immunoglobulin domains, CH1, CH2, and CH3 (for IgA,
IgD, IgG) or four immunoglobulin domains, CH1, CH2, CH3, and CH4
(for IgE and IgM). The light chain constant region comprises one
immunoglobulin domain, CL. An antibody can have the structure of an
IgA, IgG, IgE, IgD and IgM as well as any subtype thereof.
Antibodies may be from any source including in particular primate
(human and non-human primate) and primatized sources. In
particular, source may include non-primate animals, in particular s
rabbits, mice, rats, wherein antibodies may be obtained by phage
display technology.
The term "monoclonal antibody" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site. The
modifier "monoclonal" indicates the character of the antibody as
being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method.
The term "chimeric antibody" generally refers to an antibody
comprising a variable region from one source or species and at
least a portion of a constant region derived from a different
source or species, usually prepared by recombinant DNA techniques.
A typical example of chimeric antibodies includes those comprising
a murine variable region and a human constant region.
The term "humanized antibody" designates antibodies from a
non-human species having one or more complementarity determining
regions (CDRs) from said non-human species and a framework region
from a human immunoglobulin molecule. Humanized antibodies may
optionally further comprise one or more framework residues derived
from the non-human species from which the CDRs were derived.
The term "human antibody" or "fully human antibody" refers to
antibodies in which the variable regions and the constant regions
of both the heavy and the light chains are all of human origin, or
substantially identical to sequences of human origin, but not
necessarily from the same antibody.
The term "isolated antibody" refers to an antibody that has been
separated from a component of its natural environment. For
instance, an isolated antibody has been purified to greater than
95% or 99% purity as determined by methods in the art (see e.g.
Flatman et al, 2007, J Chromatogr B Analyt Technol Biomed Life Sci,
848: 79-87) including electrophoretic (e.g. SDS-PAGE, isoelectric
focusing, capillary electrophoresis) or chromatographic (e.g. ion
exchange or reverse phase HPLC (high performance liquid
chromatography) methods.
The terms "polynucleotide" or "nucleic acid molecule" refers to a
polymer comprising nucleotides. Examples of nucleic acid molecules
include DNA, RNA, locked nucleic acid (LNA), complementary DNA
(cDNA).
The term "epitope" includes any antigenic determinant capable of
specific binding to an antibody or antigen binding fragment
thereof. An epitope is a region of an antigen that is bound by an
antibody. Some epitopes comprise discontinuous sections of the
antigen's amino acid sequence, where non-contiguous amino acids are
positioned close to each other's by the spatial configuration of
the antigen ("conformational epitopes") or comprise a section of
contiguous amino acids on the antigen's amino acid sequence
("linear epitopes").
As used herewith the term "bind" or "binding" of an antibody to a
target antigen means an at least temporary interaction or
association of said antibody with, or to, said target antigen (such
as POSTN fragments) or with, or to, fragments of said target
antigen comprising an epitope recognized by said antibody.
The terms "selectively binds", "specifically binds", "specific
for", when applied to an antibody, indicate that the antibody
preferentially recognizes and/or binds the target polypeptide or
epitope, i.e. with a higher affinity than to any other antigen or
epitope, i.e. the binding to the target polypeptide can be
discriminated from non-specific binding to other antigens. The
binding affinity of an antibody can be readily determined by one of
ordinary skill in the art, for example, by Scatchard analysis
(Scatchard et al., 1949, Ann. N.Y. Acad. 1949. 51, 660-672).
As used herein, "binding affinity" generally refers to the apparent
association constant or "Ka". The Ka is the reciprocal of the
dissociation constant "Kd". Binding affinity may be determined by a
variety of methods including equilibrium dialysis, equilibrium
binding, gel filtration, ELISA, surface plasmon resonance or
spectroscopy (e.g. using a fluorescence assay). Exemplary
conditions for evaluating binding affinity are in TRIS-buffer (50
mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2 at pH 7.5). These techniques
can be used to measure the concentrations of bound and free binding
protein as a function of binding protein (or target) concentration.
The concentration of bound binding protein ([Bound]) is related to
the concentration of free binding protein ([Free]) and the
concentration of binding sites for the binding protein on the
target where (N) is the number of binding sites per target molecule
by the following equation:
[Bound]=N.times.([Free])/((1/Ka)+[Free]). Comparison of affinity
between two antibodies can be established without actually
determining the Ka value for each antibody, but based on a
quantitative measurement of affinity (e.g. by ELISA or FACS
analysis) that is proportional to Ka or a qualitative measurement
of affinity or an inference of affinity (e.g. in functional assay
or in vitro or in vivo assay).
The term "solid matrix" includes any solid phase support suitable
for carrying out an immunoassay or a method according to the
invention. It includes beads, microparticles, nanoparticles, tubes,
fabrics or plates, films, slides, wells, formed from or coated with
glass, polystyrene, polypropylene, nitrocellulose, quartz, ceramic,
dextran or other materials. For example, the solid matrix is in a
form of microtiter wells, such as a 96-well microtiter plate or 386
wells plate. Solid matrix can be coated with coupling agent such as
avidine and steptavidin.
The expression "kit" comprises at least one antibody according to
the invention or a variant thereof or a combination thereof as
described herein to be coupled or already coupled to a solid matrix
and optionally instructional material.
As used herein a "metabolic bone disease or disorder" refers to any
disease or disorder caused by abnormalities of minerals such as
calcium, phosphorus, magnesium, vitamin D, parathyroid hormone
(PTH) function or rare genetic mutations. Examples of metabolic
bone disease or disorder include, but not limited to osteoporosis,
osteomalacia (adults), rickets (children), osteitis fibrosa
cystica, Paget's disease of bone, osteoclastogenesis dysfunction,
osteopetrosis and sclerosing bone dysplasias, renal osteodystrophy,
fibrous dysplasia, cancer induced bone diseases, primary
hyperparathyroidism diseases, juvenile idiopathic osteoporosis and
van Buchem disease.
As used herein "osteoporosis" refers to a disease where decreased
bone strength increases the risk of a broken bone. It occurs due to
the imbalance between bone resorption and formation. It is further
characterised in a loss of bone mineral density and alterations in
bone quality (microstructure and material properties) leading to
fragility fractures.
The term "osteoclastogenesis dysfunction" as used herein refers to
any condition related to increased bone turnover or bone resorption
of secondary cause such a Paget's, bone loss resulting from
immobilization, osteolytic bone metastasis, bone tumours,
glucocorticoid-induced bone loss, hyperparathyroidism,
hyperthyroidism and diabetes.
The term "subject" as used herein refers to mammals. For example,
mammals contemplated by the present invention include human,
primates, domesticated animals such as dogs, cats, cattle, sheep,
pigs, horses, laboratory rodents and the like.
The term "patient" refers to a subject with a metabolic bone
disease or disorder, such as osteoporosis.
The term "patient/subject at risk of osteoporosis" refers to a
subject at risk of developing osteoporosis as confirmed e.g. by
chemical biomarkers or by measuring the bone mineral density.
As used herein, "treatment" and "treating" and the like generally
mean obtaining a desired pharmacological and physiological effect.
The effect may be prophylactic in terms of preventing or partially
preventing a disease, symptom or condition thereof and/or may be
therapeutic in terms of a partial or complete cure of a disease,
condition, symptom or adverse effect attributed to the disease. The
term "treatment" as used herein covers any treatment of a metabolic
bone disease or disorder in a mammal, particularly a human, and
includes any use of any drug that targets osteoclasts or
osteoblasts functions, including drugs targeting CatK metabolic
pathway, e.g. CatK inhibitors, or drugs targeting sclerostin. In
particular treatment of a metabolic bone disease or disorder in a
mammal, particularly a human, may include administration of POSTN
peptidic fragments.
The term "efficacy" of a treatment or method according to the
invention can be measured based on changes in the course of disease
or condition in response to a use or a method according to the
invention. For example, the efficacy of a treatment or method
according to the invention can be measured by its impact on signs
or symptoms of illness. A response is achieved when the patient
experiences partial or total alleviation, or reduction of unwanted
symptoms of illness.
The term "effective amount" as used herein refers to an amount of
at least one POSTN peptidic fragment, or a pharmaceutical
formulation thereof, that elicits a detectable reduction of the
symptoms of the disease in a subject that is being administered
said POSTN peptidic fragment, these symptoms can include, for
instance increase in bone density.
POSTN Fragments
According to one aspect of the invention are provided POSTN
peptidic fragments obtainable after CatK-dependent degradation of
POSTN.
In another aspect of the invention are provided POSTN peptidic
fragments obtainable after CatK-dependent degradation of human
POSTN, in particular human recombinant POSTN.
According to one aspect of the invention are provided POSTN
fragments obtained by an in vitro method of CatK-dependent
degradation.
In a particular embodiment, is provided an isolated peptide
comprising or consisting of an amino acids sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, or variants thereof.
In a particular embodiment, is provided an isolated peptide
consisting of 6 to 20 amino acid, comprising or consisting of an
amino acids sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or variants thereof.
In a particular embodiment, is provided an isolated peptide with a
molecular weight ranging from about 600 to about 2400 Da,
comprising or consisting of an amino acids sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, or variants thereof.
In a particular embodiment, is provided an isolated peptide
consisting of 6 to 20 amino acid with a molecular weight ranging
from about 600 to about 2400 Da, comprising or consisting of an
amino acids sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or variants thereof.
In a further embodiment, is provided an isolated peptide comprising
or consisting of an amino acids sequence selected from the group
consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID
NO: 5, or variants thereof.
In a further embodiment, is provided an isolated peptide comprising
an amino-acid sequence of SEQ ID NO: 2.
In a further embodiment, is provided an isolated peptide consisting
of an amino-acid sequence selected from the group consisting of:
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
In a further embodiment, is provided an isolated peptide having at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98% or at least 99% identity or homology
with a sequence of amino acids selected from the group consisting
of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
According to one aspect of the invention, is provided an isolated
peptide having at least 80% identity or homology with a sequence of
amino acids selected from the group consisting of: SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
In another further embodiment, is provided an isolated peptide
according to the invention having an amino acid sequence consisting
of SEQ ID NO: 2, or variant thereof.
In another further embodiment, is provided an isolated peptide
according to the invention having an amino acid sequence, wherein
at least one amino acid is added or deleted at the C-terminal
end.
In another further embodiment, is provided an isolated peptide
according to the invention having an amino acid sequence, wherein
at least two amino acids are added or deleted at the C-terminal
end.
Methods of Producing POSTN Fragments
Synthetic chemistry methods, such as solid-phase peptide synthesis,
can be used to synthesize the polypeptides according to the
invention. Purification of those peptides may be carried out by
means of any technique known in the art of protein/peptide
purification. Exemplary techniques include ion-exchange
chromatography, hydrophobic interaction chromatography, and
immunoaffinity methods.
According to an embodiment, the invention provides a process of
producing POSTN fragments or variants thereof according to
invention comprising incubating POSTN of with CatK, in particular
human CatK, such as human recombinant CatK.
According to an embodiment, the invention provides a process of
producing POSTN fragments or variants thereof according to
invention comprising incubating POSTN of with CatK, wherein POSTN
and CatK are present at a ratio ranging from about 100:1 to about
22:1, wherein incubation process is performed at a temperature from
about 19.degree. to about 38.degree., wherein time of a reaction is
from about 1 minute to about 24 h, and wherein said reaction is
performed at pH from about 4 to about 6.
It is understood that both POSTN and CatK can be synthetic or
derived from any species, in a preferred embodiment POSTN and CatK
are human POSTN and CatK.
According to an embodiment, the invention provides a process of
producing POSTN fragments or variants thereof according to
invention comprising incubating human recombinant POSTN with CatK
of SEQ ID NO: 19.
According to another embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK at a ratio ranging
from about 100:1 to about 22:1.
The process of producing POSTN fragments or variants thereof
according to the invention comprising incubating human recombinant
POSTN with CatK at a ratio of 50:1.
The process of producing POSTN fragments or variants thereof
according to the invention comprising incubating human recombinant
POSTN with CatK at temperature from about 19 to about 38.degree.
C.
The process of producing POSTN fragments or variants thereof
according to the invention comprising incubating human recombinant
POSTN with CatK at temperature from about 35 to about 38.degree.
C.
The process of producing POSTN fragments or variants thereof
according to the invention comprising incubating human recombinant
POSTN with CatK at a temperature of about 37.degree. C.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK from about 1 minute to
about 24 h.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK from about 1 minute to
about 5 h.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK from about 10 minutes
to about 60 minutes.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK from about 10 minutes
to about 30 minutes.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK at pH from about 4 to
about 6.
According to further embodiment, the process of producing POSTN
fragments or variants thereof according to the invention comprising
incubating human recombinant POSTN with CatK at pH of about
5.5.
POSTN Fragments Binding Antibodies
In one embodiment of the invention are provided isolated antibodies
or antigen-binding fragments thereof specific for POSTN fragments
according to the invention.
In one embodiment of the invention are provided isolated antibodies
specific for POSTN fragments of the invention.
In an alternative embodiment of the invention are provided isolated
antibodies or antigen-binding fragments thereof, specific for POSTN
fragments of the invention, in particular human POSTN fragments as
described herewith.
In an alternative embodiment of the invention are provided isolated
antibodies or antigen-binding fragments thereof, specific for POSTN
fragments, in particular human POSTN fragments, further
characterized by their binding to an epitope on POSTN fragments, as
described herewith.
The protein to which the antibodies according to the invention or
fragments thereof, bind to POSTN fragments of the invention of any
species.
The antibodies according to the present invention generally exhibit
a high specificity for human POSTN fragments of the invention.
In a further embodiment, are provided isolated antibodies specific
for a peptide of 6 to 20 amino acids comprising or consisting in an
amino-acid sequence of SEQ ID NO: 2.
In a particular embodiment, the antibodies according to the
invention, or fragments thereof, bind preferentially to POSTN
fragments of the invention and exhibit a weak, or virtually no
(i.e. negligible or not detectable), binding to full POSTN.
It is understood that any variant of an antibody according to the
invention, or fragment thereof, that is described herewith is able
to bind POSTN fragments of the invention. In a particular
embodiment, such variant can show the same or even higher binding
affinity for POSTN fragments of the invention, in comparison to the
parental antibody or fragment from which said variant derives.
The antibodies according to the invention can be monoclonal
antibodies, polyclonal antibodies, human antibodies, humanized
antibodies, chimeric antibodies, and further engineered antibodies
as long as the characteristic properties of the invention are
retained, in particular the ability of binding to the target
antigen, more specifically to the POSTN fragments of the
invention.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof which specifically bind to POSTN fragments, are
monoclonal antibodies.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof specifically bind to a POSTN fragment comprising
or consisting of an amino acid sequence selected from the group
consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or
variants thereof.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof specifically bind to a POSTN fragment consisting
of 6 to 20 amino acid, comprising or consisting of an amino acid
sequence selected from the group consisting of: SEQ ID NO: 2, SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18, or variants thereof.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof specifically bind to a POSTN fragment with a
molecular weight ranging, from about 600 to about 2400 Da
comprising or consisting of an amino acid sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, or variants thereof.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof specifically bind to a POSTN fragment consisting
of 6 to 20 amino acid with a molecular weight ranging from about
600 to about 2400 Da comprising or consisting of an amino acid
sequence selected from the group consisting of: SEQ ID NO: 2, SEQ
ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18, or variants thereof.
In a preferred embodiment of the invention, the antibodies specific
for POSTN fragments according to the invention, or fragments
thereof specifically bind to peptide consisting or comprising an
amino acid sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
In a more preferred embodiment of the invention, the antibodies
specific for POSTN fragment according to the invention, or
fragments thereof specifically bind to a peptide of an amino acid
sequence consisting in SEQ ID NO: 2.
Antibodies can be produced by standard techniques in the field
including the recovery of polyclonal antibodies from the serum of
laboratory or farm animals, or eggs from chicken which have been
injected with the antigen of interest, the recovery of monoclonal
antibodies produced by fusing antibody-secreting spleen cells from
immunized mice, or rats, or rabbits with immortal myeloma cell to
create monoclonal hybridoma cell lines that express the specific
antibody in cell culture supernatant. Alternatively, recombinant
antibodies can be produced in various host cells including
mammalian cells, plant cells, bacteria, yeasts, which have been
engineered to express said antibodies.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof are produced by injecting synthetic POSTN
fragment to rabbits and collecting serum thereof at defined
intervals.
In a particular embodiment of the invention, the antibodies
specific for POSTN fragments according to the invention, or
fragments thereof are produced by phase-display technology after
immunization of camelides.
In another aspect of the invention the POSTN fragments or variants
thereof can be used as an immunogen for generating antibodies
according to the invention.
The immunogens for generating antibodies according to the invention
comprise a peptide comprising an amino acids sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, or variants thereof.
The immunogens for generating antibodies according to the invention
comprise a peptide consisting of 6 to 20 amino acid, comprising an
amino acids sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or variants thereof.
The immunogens for generating antibodies according to the invention
comprise a peptide with a molecular weight ranging from about 600
to about 2400 Da, comprising an amino acids sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID
NO: 18, or variants thereof.
The immunogens for generating antibodies according to the invention
comprise a peptide consisting of 6 to 20 amino acid with a
molecular weight ranging from about 600 to about 2400 Da,
comprising an amino acids sequence selected from the group
consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or
variants thereof.
According to a particular aspect, an immunogen for generating
antibodies according to the invention is a peptide of 6 to 20 amino
acids or consisting in an amino acid sequence of SEQ ID NO: 2.
Preferably, the immunogen for generating antibodies according to
the invention is a peptide of an amino acid sequence selected from
the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4
and SEQ ID NO: 5, or variants thereof.
In another aspect of the invention, the isolated antibodies or
fragment thereof according to the invention are optionally
conjugated to an accessory molecule, and are then also referred to
herein as "conjugated antibodies or "conjugated antibody
fragments".
The accessory molecule may be conjugated to the antibody or
antibody fragment directly or via a spacer of suitable length for
instance as described in Kellogg et al., 2011, Bioconjug Chem, 22:
717-27).
In one embodiment, particularly adapted for diagnostic purposes,
the accessory molecule can be, for example, a coupling agent such
as biotin, a labeling group including radioisotopes (e.g. 3H, 14C,
32P, 35S, 125I), chromogenic labels, e.g. enzymes which can be used
to convert a substrate to a detectable colored (e.g. horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase) or
fluorescent compound (e.g. Green Fluorescent Protein, Red
Fluorescent Protein), spectroscopic labels (e.g. fluorescent labels
such as fluorescein and its derivatives like FITC, Texas red,
cyanine dyes, photocyan, rhodamine, or labels presenting a visible
color), luminescent labels including luciferins, affinity labels
which may be developed by a further compound specific for the label
and allowing easy detection and quantification, or any other label
used in standard ELISA.
According to another embodiment, is provided a use of the
antibodies according to the invention for the coating of a solid
matrix for performing an immunoassay.
Nucleic Acids Encoding the Polypeptides of the Invention
According to one embodiment, is provided an isolated nucleic acid
molecule encoding a peptide or variants thereof according to the
invention.
According to one embodiment, is provided an isolated nucleic acid
molecule encoding a POSTN fragments or variants thereof according
to the invention.
The isolated nucleic acid according to the invention may be, for
instance, natural DNA or RNA or a recombinant or synthetic DNA, RNA
or LNA or a recombinant nucleic acid molecule comprising any of the
nucleic acid molecules according to the invention either alone or
in combination.
Nucleic Acids Encoding the Antibodies of the Invention
According to another embodiment, is provided an isolated nucleic
acid molecule encoding an antibody or antigen-binding fragment
thereof according to the invention.
In a particular embodiment, it is provided an isolated nucleic acid
comprising a nucleic acid sequence encoding an antibody or
antigen-binding fragment thereof according to the invention.
Vectors and Host Cells for Production and Purification of the
Polypeptides of the Invention
In one embodiment, the invention provides a recombinant expression
vector comprising a nucleic acid molecule according to the
invention, wherein the vector optionally comprises an expression
control sequence, allowing expression in prokaryotic or eukaryotic
host cells of the encoded polypeptide, operably linked to said
nucleic acid molecule.
Numerous expression systems can be used, including without
limitation chromosomes, episomes, and derived viruses. More
particularly, the recombinant vectors used can be derived from
bacterial plasmids, transposons, yeast episomes, insertion
elements, yeast chromosome elements, viruses such as baculovirus,
papilloma viruses such as SV40, vaccinia viruses, adenoviruses, fox
pox viruses, pseudorabies viruses, retroviruses. These recombinant
vectors can equally be cosmid or phagemid derivatives.
The nucleic acid sequence can be inserted in the recombinant
expression vector by methods well known to a person skilled in the
art such as, for example, those that are described in MOLECULAR
CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.
The recombinant vector can include nucleotide sequences that allow,
control or regulate the expression and the transcription of a
polynucleotide of the invention as well as the translation of a
polypeptide of the invention, these sequences being selected
according to the host cells that are used.
Thus, for example, an appropriate secretion signal can be
integrated in the recombinant vector so that the polypeptide,
encoded by the nucleic acid molecule of the invention, will be
directed towards the lumen of the endoplasmic reticulum, towards
the periplasmic space, on the membrane or towards the extracellular
environment. The choice of an appropriate secretion signal may
facilitate subsequent protein purification.
In a further embodiment, it is provided a host cell comprising a
recombinant vector according to the invention.
The introduction of the recombinant vector in a host cell can be
carried out according to methods that are well known to a person
skilled in the art, such as those described in BASIC METHODS IN
MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-Hill Professional
Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL,
supra, such as transfection by calcium phosphate, transfection by
DEAE dextran, transfection, microinjection, transfection by
cationic lipids, electroporation, transduction or infection.
The host cell can be, for example, bacterial cells such as E. coli
or Streptomyces, cells of fungi such as Aspergillus and yeasts such
as Saccharomyces, insect cells, Chinese Hamster Ovary cells (CHO),
C127 mouse cell line, BHK cell line of Syrian hamster cells, Human
Embryonic Kidney 293 (HEK 293) cells. In a particular embodiment,
the host cell is a CHO cell or a HEK 293 cell.
The host cells can be used, for example, to express a polypeptide
of the invention. After purification by standard methods, the
polypeptide of the invention can be used in a method described
hereinafter.
For instance, when expression systems that secrete the recombinant
protein are employed, the culture medium may first be concentrated
using a commercially available protein concentration filter, for
example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate can be applied to
a purification matrix such as a gel filtration matrix.
Alternatively, an anion exchange and/or an affinity resin can be
employed. The matrices can be acrylamide, agarose, dextran,
cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Finally, one
or more reversed-phase high performance liquid chromatography
(RP-HPLC) steps employing hydrophobic RP-HPLC media can be employed
to further purify the antibodies or fragments thereof. Some or all
of the foregoing purification steps, in various combinations, are
well known and can be employed to provide a substantially
homogeneous recombinant protein.
Recombinant protein produced in bacterial culture can be isolated
by initial disruption of the host cells, centrifugation, extraction
from cell pellets if an insoluble polypeptide, or from the
supernatant fluid if a soluble polypeptide, followed by one or more
concentration, salting-out, ion exchange, affinity purification or
size exclusion chromatography steps. Microbial cells can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing
agents.
In another embodiment, the invention provides a process for
producing cells capable of expressing a polypeptide according to
the invention, comprising genetically engineering cells with a
vector or a nucleic acid according to the invention.
In another embodiment, the invention provides a process for
producing peptides according to the invention comprises culturing a
host cell transformed with an expression vector comprising a
nucleic sequence that encodes said antibodies or fragments thereof
under conditions sufficient to promote expression of said
polypeptides. The peptides according to the invention are then
recovered from culture medium or cell extracts, depending upon the
expression system employed.
In another embodiment, the invention provides a process for
producing antibodies or fragments thereof according to the
invention comprises culturing a host cell transformed with an
expression vector comprising a nucleic sequence that encodes said
antibodies or fragments thereof under conditions sufficient to
promote expression of said polypeptides. The antibody or fragment
thereof according to the invention is then recovered from culture
medium or cell extracts, depending upon the expression system
employed. As known to the skilled artisan, procedures for purifying
a recombinant protein will vary according to such factors as the
type of host cells employed and whether or not the recombinant
protein is secreted into the culture medium as described above.
Compositions
The invention provides pharmaceutical or therapeutic agents as
compositions and methods for treating a patient, preferably a
mammalian patient, and most preferably a human patient who is
suffering from a medical disorder, and in particular a metabolic
bone disorder. Alternatively, the invention provides methods for
preventing a medical disorder, and in particular a metabolic bone
disorder.
In one embodiment, is provided a pharmaceutical composition
comprising one or more of (i) peptide according to the invention or
fragment thereof, (ii) a nucleic acid according to the invention,
(iii) a vector according to the invention, and/or (iv) a host cell
according to the invention, and at least one pharmaceutically
acceptable carrier. Pharmaceutical compositions of the invention
can contain one or more peptide of the invention or fragment
thereof in any form described herein.
Compositions of this invention may further comprise one or more
pharmaceutically acceptable additional ingredient(s) such as alum,
stabilizers, antimicrobial agents, buffers, coloring agents,
flavoring agents, adjuvants, and the like.
The compounds of the invention, together with a conventionally
employed adjuvant, carrier, diluent or excipient may be placed into
the form of pharmaceutical compositions and unit dosages thereof,
and in such form may be employed as solids, such as tablets or
filled capsules, freeze-dried forms, or liquids such as solutions,
suspensions, emulsions, elixirs, or capsules filled with the same,
all for oral use, or in the form of sterile injectable solutions
for parenteral (including subcutaneous) use. Such pharmaceutical
compositions and unit dosage forms thereof may comprise ingredients
in conventional proportions, with or without additional active
compounds or principles, and such unit dosage forms may contain any
suitable effective amount of the active ingredient commensurate
with the intended daily dosage range to be employed.
Compositions of this invention may be liquid formulations
including, but not limited to, aqueous or oily suspensions,
solutions, emulsions, syrups, and elixirs. Liquid forms suitable
for oral administration may include a suitable aqueous or
non-aqueous vehicle with buffers, suspending and dispensing agents,
colorants, flavors and the like. The compositions may also be
formulated as a dry product for reconstitution with water or other
suitable vehicle before use. Such liquid preparations may contain
additives including, but not limited to, suspending agents,
emulsifying agents, non-aqueous vehicles and preservatives.
Suspending agent include, but are not limited to, sorbitol syrup,
methylcellulose, glucose/sugar syrup, gelatin, hydroxyethyl
cellulose, carboxymethyl cellulose, aluminum stearate gel, and
hydrogenated edible fats. Emulsifying agents include, but are not
limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous
vehicles include, but are not limited to, edible oils, almond oil,
fractionated coconut oil, oily esters, propylene glycol, and ethyl
alcohol. Preservatives include, but are not limited to, methyl or
propyl p-hydroxybenzoate and sorbic acid. Further materials as well
as processing techniques and the like are set out in Part 5 of
Remington's The Science and Practice of Pharmacy, 22.sup.nd
Edition, 2012, Pharmaceutical Press and the University of the
Sciences, Philadelphia College of Pharmacy, which is incorporated
herein by reference.
Solid compositions of this invention may be in the form of tablets
or lozenges formulated in a conventional manner. For example,
tablets and capsules for oral administration may contain
conventional excipients including, but not limited to, binding
agents, fillers, lubricants, disintegrants and wetting agents.
Binding agents include, but are not limited to, syrup, accacia,
gelatin, sorbitol, tragacanth, mucilage of starch and
polyvinylpyrrolidone. Fillers include, but are not limited to,
lactose, sugar, microcrystalline cellulose, maizestarch, calcium
phosphate, and sorbitol. Lubricants include, but are not limited
to, magnesium stearate, stearic acid, talc, polyethylene glycol,
and silica. Disintegrants include, but are not limited to, potato
starch and sodium starch glycollate. Wetting agents include, but
are not limited to, sodium lauryl sulfate. Tablets may be coated
according to methods well known in the art.
Injectable compositions are typically based upon injectable sterile
saline or phosphate-buffered saline or other injectable carriers
known in the art.
Compositions of this invention may also be formulated as
transdermal formulations comprising aqueous or non-aqueous vehicles
including, but not limited to, creams, ointments, lotions, pastes,
medicated plaster, patch, or membrane.
Compositions of this invention may also be formulated for
parenteral administration including, but not limited to, by
injection or continuous infusion. Formulations for injection may be
in the form of suspensions, solutions, or emulsions in oily or
aqueous vehicles, and may contain formulation agents including, but
not limited to, suspending, stabilizing, and dispersing agents. The
composition may also be provided in a powder form for
reconstitution with a suitable vehicle including, but not limited
to, sterile, pyrogen-free water.
Compositions of this invention may also be formulated as a depot
preparation, which may be administered by implantation or by
intramuscular injection. The compositions may be formulated with
suitable polymeric or hydrophobic materials (as an emulsion in an
acceptable oil, for example), ion exchange resins, or as sparingly
soluble derivatives (as a sparingly soluble salt, for example).
The compounds of this invention can also be administered in
sustained release forms or from sustained release drug delivery
systems. A description of representative sustained release
materials can also be found in the incorporated materials in
Remington's Pharmaceutical Sciences.
Injectable formulations are particularly appropriate for
administering the compositions according to the invention.
In another embodiment, the invention provides an imaging
composition or diagnostic composition comprising an antibody or an
antigen-binding fragment thereof specific for at least one peptide
of the invention or variant thereof as described herewith.
In another embodiment, the invention provides a diagnostic
composition comprising an antibody or antigen-binding fragment
thereof specific for a peptide of the invention or variant thereof
as described herewith for detection of a peptide of the invention
or variants thereof.
In one embodiment, diagnostic composition according to the
invention comprise an antibody or antigen-binding fragment thereof
specific for a peptide of the invention or variant thereof as
described herewith conjugated to a moiety selected from the group
consisting of a radioisotope, a biotin, an avidin, a strepavidin, a
chromophore, a fluorophore, a chemiluminescent moiety, a hapten and
an enzyme.
The imaging composition or diagnostic composition according to the
invention is useful for detecting elevated levels of a peptide of
the invention or variant thereof associated with metabolic bone
diseases.
Combination
According to the invention, peptides or variant thereof according
to the invention can be administered alone or in combination with a
co-agent useful in the prevention and/or treatment of an metabolic
bone disorder, for example immune modulatory drugs including
biologics, small molecules, and vaccines.
Peptides of the invention or variant thereof can be administered to
an individual prior to, simultaneously or sequentially with other
therapeutic regimens or co-agents useful in the prevention and/or
treatment of a metabolic bone disease. The peptides of the
invention or variant thereof according to the invention that are
administered simultaneously with said co-agents can be administered
in the same or different compositions and in the same or different
routes of administration.
Mode of Administration
Compositions of this invention may be administered in any manner
including, but not limited to, orally, parenterally, sublingually,
transdermally, rectally, transmucosally, topically, via inhalation,
via buccal or intranasal administration or intra bladder, or
combinations thereof.
Parenteral administration includes, but is not limited to,
intravenous, intra-arterial, intra-peritoneal, subcutaneous,
intramuscular, intra-thecal, and intra-articular. The compositions
of this invention may also be administered in the form of an
implant, which allows slow release of the compositions as well as a
slow controlled i.v. infusion.
In a particular embodiment, peptides of the invention or variant
thereof according to the invention are administered systemically or
locally.
In a particular embodiment, a peptide of the invention or variant
thereof according to the invention is administered by subcutaneous
or intravenous route.
The dosage administered, as single or multiple doses, to an
individual will vary depending upon a variety of factors, including
pharmacokinetic properties, patient conditions and characteristics
(sex, age, body weight, health, size), extent of symptoms,
concurrent treatments, frequency of treatment and the effect
desired.
Methods According to the Invention
In another aspect, the invention provides an immunoassay
preparation comprising at least one antibody according to the
invention.
According to a particular aspect, an immunoassay preparation
according to the invention can be used in a method for the
detection of POSTN fragments, such as a method of measuring
detection of POSTN fragments contained in a sample.
Examples of a method utilizing an antigen-antibody reaction in
detection of POSTN fragments contained in a sample include enzyme
immunoassays (ELISA and EIA), fluoroimmunoassays (FIAs),
radioimmunoassays (RIAs), luminescence immunoassays (LIAs), enzyme
antibody techniques, fluorescence antibody techniques,
immunochromatographies, immunonephelometries, latex nephelometries,
latex agglutination assays, erythrocyte agglutination assays,
particle agglutination assays, the method described in, for
example, Japanese Patent Laid-Open No. H09-229936 or Japanese
Patent Laid-Open No. H10-132819 using a carrier having a surface
onto which a substance that specifically binds to a substance to be
measured (analyte) is immobilized so as to cover the surface and
using particles onto which a substance that specifically binds to
the substance to be measured (analyte) is immobilized, and the
enzyme-linked ligandsorbent assay (ELSA) described by Dahlbeack et
al., 1998, Thromb. Haemost., 79, 767-772; WO 98/23963).
According to an embodiment, the invention provides a method for
detecting a POSTN fragment from a biological fluid sample of a
mammalian subject comprising the steps of: (a) providing a
biological fluid sample obtained from a mammalian subject; (b)
bringing the said biological fluid sample into contact with a solid
matrix where at least one antibody is bound to, wherein the
contacting is under conditions sufficient for binding a POSTN
fragment present in the said biological fluid sample to the said at
least one antibody through antigen-antibody interactions and
wherein the said at least one antibody is specific for POSTN
fragment or any variant thereof; (c) removing the biological fluid
sample from the solid matrix for removing any unbound POSTN
fragment from the surface of the said solid matrix; (d) detecting
the presence of an antigen-antibody complex bound to the said solid
matrix, wherein the presence of the said complex is indicative that
the biological fluid sample contains one or more POSTN
fragments.
According to an embodiment, the invention provides a method for
detecting a POSTN fragment from a biological fluid sample of a
mammalian subject comprising the steps of: (a) providing a
biological fluid sample obtained from a mammalian subject; (b)
bringing said biological fluid sample into contact with at least
one antibody, wherein the contacting is under conditions sufficient
for binding a POSTN fragment of the invention present in said
biological fluid sample to said at least one antibody through
antigen-antibody interactions and wherein said at least one
antibody is specific for POSTN fragment of the invention or any
variant thereof; (c) bringing sample obtained under step b) into
contact with a solid matrix where at least one POSTN fragment of
the invention is bound to, wherein the contacting is under
conditions sufficient for binding an antibody specific for POSTN
fragment of the invention present in said sample to said at least
one POSTN fragment of the invention through antigen-antibody
interactions; (d) washing the solid matrix for removing any unbound
antibody from the surface of the said solid matrix; (e) detecting
the presence of an antigen-antibody complex bound to the said solid
matrix, wherein the presence of the said complex is indicative that
the biological fluid sample contains one or more POSTN fragments of
the invention.
In another aspect, the invention provides a method for detecting a
POSTN fragment from a biological fluid sample of a mammalian
subject comprising the steps of: (a) providing a biological fluid
sample obtained from a mammalian subject; (b) providing a solid
support having bound thereto at least one POSTN fragments of the
invention; (c) bringing said solid support into contact with said
biological fluid sample; (d) bringing said solid support into
contact with at least one antibody specific for POSTN fragment of
the invention or any variant thereof, wherein the contacting is
under conditions sufficient for binding a POSTN fragment of the
invention present in said biological fluid sample to said at least
one antibody through antigen-antibody interactions; (e) washing the
solid matrix for removing any unbound antibody from the surface of
the said solid matrix; (f) detecting the presence of an
antigen-antibody complex bound to the said solid matrix, wherein
the presence of the said complex is indicative that the biological
fluid sample contains one or more POSTN fragments of the
invention.
According to an embodiment, a solid support having bound thereto at
least one POSTN fragment of the invention can be obtained by
coating a microtiter plate with at least one POSTN fragment of the
invention or by modifying a surface of a microtiter plate to be
covalently conjugated to at least one POSTN fragment of the
invention. For example, biotinylated peptides of the invention can
be incubated on streptavidin pre-coated microplates according to
standard methods.
According to an embodiment, the detection of the presence of an
antigen-antibody complex bound to the solid matrix can be achieved
with a secondary antibody. According to an embodiment, a method for
detecting a POSTN fragment according to the invention is indicative
of a metabolic bone disorder in said subject.
According to an embodiment, a method for detecting a POSTN fragment
according to the invention is indicative of the effect of treatment
of a metabolic bone disorder in said subject.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide comprising or consisting in sequence of amino acid
selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide consisting of 6 to 20 amino acid, comprising a or
consisting in sequence of amino acid selected from: SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide with a molecular weight ranging from about 600 to
about 2400 Da, comprising a or consisting in sequence of amino acid
selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide consisting of 6 to 20 amino acid with a molecular
weight ranging from about 600 to about 2400 Da, comprising a or
consisting in sequence of amino acid selected from: SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17 and SEQ ID NO: 18.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide comprising or consisting in a sequence of amino acids
selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID
NO: 5.
According to a further embodiment, is provided a method according
to the invention, wherein said at least one antibody is specific
for a peptide comprising or consisting in a sequence of amino acids
of SEQ ID NO: 2.
According to another further embodiment, is provided a method
according to the invention, wherein the said biological fluid
sample is brought into contact with the said solid matrix under
step b), where at least one antibody or variant thereof according
to the invention is bound to said solid matrix.
According to another further embodiment, is provided a method
according to the invention, wherein a known quantity of capture
antibody is bound to said solid matrix under step b).
According to another further embodiment, is provided a method for
detecting a POSTN fragments from a biological fluid sample of a
mammalian subject wherein the said biological fluid sample is
brought into contact with the said solid matrix under step b),
where a combination of antibodies or of variants thereof is bound
to the said solid matrix and where the combination comprises: a)
one antibody specific for a peptide having a sequence of amino
acids of SEQ ID NO: 2 or a variant thereof; and b) at least one
antibody specific for a peptide having an amino acid sequence
selected from: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 or a variant
thereof.
According to another further embodiment, is provided a method for
detecting a POSTN fragments from a biological fluid sample of a
mammalian subject wherein the said biological fluid sample is
brought into contact with the said solid matrix under step b),
where a combination of antibodies or of variants thereof is bound
to the said solid matrix and where the combination comprises: a)
one antibody specific for a peptide having a sequence of amino
acids of SEQ ID NO: 2; and b) at least one antibody specific for a
peptide having a sequence of amino acids selected from: SEQ ID NO:
3, SEQ ID NO: 4 and SEQ ID NO: 5.
According to another further embodiment, is provided a method
according to the invention, wherein the method further comprises a
step of comparing the signal obtained under the detection step d)
with the same signal obtained for at least one control sample,
wherein the signal obtained for the said at least one control
sample is collected previously, simultaneously or posteriori to the
detection step d) for the said biological fluid sample.
Detection of the captured/bound POSTN fragments under step d) by
any suitable method known in the art for detecting captured
antibodies or proteins on surfaces such as optical detection (e.g.
ELISA), mass variation detection (e.g. surface Plasmon resonance,
mass spectrometry), electrical detection (e.g. impedance
spectroscopy, electrochemical) techniques. Results of the assay may
be qualitative or quantitative. The amount of POSTN fragments
associated with antibodies can be compared with positive and
negative controls. The controls are typically run concomitantly
with the sample to be tested. A positive control can be a serum or
a solution containing a POSTN fragment that is immunoreactive with
at least one antibody according to the invention. A negative
control can be a serum or solution which does not contain a POSTN
fragment that is immunoreactive with at least one antibody
according to the invention. For quantization, a calibration curve
using known quantities of POSTN fragments to at least one antibody
according to the invention can be generated and/or used.
The comparison with normal healthy biological fluid samples may be
achieved with different methods. According to one embodiment, it
may be carried out by including a control reaction with a blood
sample from healthy subject. According to another embodiment, it
may be carried out by employing a value for the concentration of
the endogeneous POSTN fragments for a typical biological fluid
sample from a healthy subject. Typically, the comparison of the
level of endogeneous POSTN fragments present in a sample under
investigation may be performed with respect to a value determined
in each single testing procedure or to a predetermined value. The
predetermined value may be determined for the testing procedure in
general, or alternatively, the value may be valid only for a
certain batch of testing reagents. For example, the reference value
may be valid for a defined calibration period only and may be
redefined upon calibration of the testing process.
According to another further embodiment, is provided a method of
monitoring the effects of a treatment of subjects with osteoporosis
comprising detection of POSTN fragments.
According to another further embodiment, is provided a method of
monitoring progression of treatment of patients with osteoporosis
comprising detection of POSTN fragments.
According to another further embodiment, is provided a method of
diagnosis of patients at risk of osteoporosis comprising detection
of POSTN fragments.
According to another further embodiment, is provided a method of
diagnosis of subjects with a metabolic bone disorder comprising
detection of POSTN fragments.
According to another further embodiment, is provided a method of
monitoring the effects of a treatment of subject with a metabolic
bone disorder comprising detection of POSTN fragments.
According to a further embodiment, are provided methods according
to the invention wherein a metabolic bone disorder is
osteoporosis.
According to a further embodiment, is provided a method according
to the invention, wherein said method is an ex vivo method.
According to another further embodiment, is provided a method for
preventing and/or treating of subjects with a metabolic bone
disorder comprising administering an isolated peptide POSTN
fragment or variants thereof.
Kit and Method Using Thereof
According to one aspect, the invention relates to a kit for
carrying out a method according to the invention.
According to one aspect, the invention provides a kit for detecting
the formation of Cat K proteolytic fragments of POSTN in the bone
cortical compartment.
According to a further aspect, the invention provides a kit
comprising (1) a solid support having bound thereto at least one
POSTN fragment of the invention; and optionally: (2) at least one
antibody according to the invention specific for said at least one
POSTN fragment; (3) at least one unbound POSTN fragment of the
invention to serve as positive control; (4) at least one detection
agent for detecting the complex that forms between said at least
one POSTN fragment of the invention present in a biological sample
and said at least one antibody according to the invention specific
for said at least one POSTN fragment.
According to a particular aspect, the invention provides an ELISA
kit, more particularly a competitive ELISA kit.
According to another aspect of the invention, is provided a kit
comprising at least one antibody according to the invention or a
variant thereof.
According to another aspect of the invention, is provided a kit for
detecting a POSTN fragment of the invention in a biological fluid
sample, the kit comprising at least one antibody according to the
invention or a variant thereof.
The kit according to the invention comprises at least one antibody
according to the invention, a variant thereof or a combination
thereof for coupling, or already coupled to a solid matrix as solid
phase support as referred herein. Various solid matrices can be
used, including but not limited to glass, polystyrene,
polypropylene, nitrocellulose, quartz, ceramic, dextran or other
materials. Suitable forms of the solid matrix include beads,
microparticles, nanoparticles, tubes, fabrics or plates, films,
slides, wells, formed from or coated with these materials.
Typically, the solid matrix comprises microtiter wells, such as a
96-well microtiter plate.
An antibody of the invention can be immobilized onto a solid-phase
carrier by adsorption and/or binding through a known method such as
physical adsorption, chemical binding, or the both.
According to another further embodiment, is provided a use of a kit
of the invention for the detection of a metabolic bone disorder in
a subject or for monitoring the course of a treatment of said
disorder in a subject.
According to another further embodiment, is provided a use of a kit
of the invention for detecting a POSTN fragment in a biological
fluid sample.
The method, the kit and uses according to the invention may be
suited for screening purposes as well as for diagnostic purposes
and may be applied in primary diagnosis as well as in monitoring of
disease course during or after treatment.
According to a particular aspect, the detection of a one or more
POSTN fragment(s) according to the invention, in particular a
fragment comprising or consisting in a sequence of a peptide
according to SEQ ID NO: 2 is a biomarker of the bone quality and is
indicative of a risk of fracture in a subject. The detection of
such fragment(s) by antibodies specific for the fragment(s) is
particularly useful in a method or a use according to the
invention.
References cited herein are hereby incorporated by reference in
their entirety. The present invention is not to be limited in scope
by the specific embodiments and drawings described herein, which
are intended as single illustrations of individual aspects of the
invention, and functionally equivalent methods and components are
within the scope of the invention. The examples illustrating the
invention are not intended to limit the scope of the invention in
any way.
EXAMPLES
Ahx (2-aminohexanoic acid), AUC (area under the curve), BSA (bovine
serum albumin), BMD (Bone mineral density), BMI (body mass index),
BV/TV (bone volume on total volume), CatK (Cathepsin K), CI
(confidence interval), Ct. (cortical bone), Ct.Th (Ct thickness),
CTX (cross-linked C-terminal telopeptide of type I collagen), CV
(coefficient of variability), DXA (dual-energy x-ray
absorptiometry), Ec (endocortical surface), FRAX (fracture risk
assessment tool), HRP (horseradish peroxidase), KLH (keyhole limpet
hemocyanin), LC-MS/MS (liquid chromatography-tandem mass
spectrometry), MI (moment of inertia), MI (moment of inertia), MW
(Molecular weight), OD (optical density), P1NP (procollagen type 1
N-terminal propeptide), POSTN (periostin), Ps (periosteum surface),
ROC (Receiver Operating Characteristic curve), RT (room
temperature), SD (standard deviation), SDS-PAGE (sodium dodecyl
sulfate-polyacrylamide gel electrophoresis), sPOSTN (serum POSTN),
STD (Standard), Tb. (trabecular bone), TRIS (Tris(hydroxymethyl)
aminomethane), TBS (Tris-buffered saline), vBMD (volumetric bone
mineral density).
Example 1
CatK Digestion of Human POSTN
To identify the human POSTN fragments resulting from CatK dependent
digestion lasting 1 h, 2 h, 3 h or overnight, polyacrylamide gel
electrophoresis was used on POSTN protein sample mixed with an
anionic detergent (sodium dodecyl sulfate) (SDS-PAGE).
CatK digestion, targeted LC-MS/MS and silver stained SDS-PAGE.
Human recombinant POSTN (1.1 .mu.M) was incubated with human CatK
of SEQ ID NO: 19 (2.24 .mu.M) at 37.degree. C. Times and POSTN/CatK
ratios are indicated for each protocol below. The inhibitor E64
(250 .mu.M) was added to stop the digestion at the end of
incubation. Then, the digests were analyzed by SDS-Page
electrophoresis. Various POSTN fragments bands were excised from
the SDS-Page gel and digested with trypsin. Then, the tryptic
peptides were concentrated and separated by reverse phase
chromatography on a PepMap100, C18, 5 .mu.m, 100 .ANG., 300
.mu.m.times.25 mm column from Dionex (ThermoScientific) using a
gradient of 5 to 40% acetonitrile, 0.1% formic acid in 60 min at
300 nL/min. Furthermore, to maximize the sequence coverage, the
analysis was conducted on 2 mass spectrometers, LTQ Velos Mass
Spectrometer (ThermoScientific) and Qstar XL Mass Spectrometer
(Applied Biosystems).
About 105 of unique POSTN fragments were observed after CatK
digestion of POSTN lasting 3 h. POSTN fragments observed after CatK
digestion corresponded to 62% of POSTN sequence. Several POSTN
fragments were characterised by overlapping sequences indicating a
non-specific type of cleavage by enzyme CatK. Two protein bands of
POSTN fragments were detected of about 35kDa and 7 kDa (FIG. 2A).
The bands were not observed in negative control where no enzyme was
added to POSTN (FIG. 2A). Digestion of constant amount of POSTN
with increasing amount of CatK indicates that the abundance ratio
of POSTN fragments increased with increasing amount of enzyme,
saturating at a ratio lower than POSTN/CatK of 11:1 (FIG. 2B).
This data show that human POSTN is a substrate to CatK-dependent
enzymatic digestion that is generating large amount of several
peptides which may serve as markers of CatK activity in
periosteum.
Example 2
Identification of POSTN Fragments
Several CatK-dependent POSTN fragments were separated and
identified with the use of targeted liquid chromatography-tandem
mass spectrometry (LC-MS/MS).
CatK digestion, targeted LC-MS/MS and silver stained SDS-PAGE as
described in Example 1. Twenty-one different POSTN fragments were
separated in a single LC-MS/MS analysis (FIG. 3A) which included
peptides 1-17 which were further analysed below and peptide 18 (SEQ
ID NO: 22), peptide 19 (SEQ ID NO: 23), peptide 20 (SEQ ID NO: 24)
and peptide 21 (SEQ ID NO: 25). The most abundant CatK-digested
POSTN fragments of SEQ ID NO: 2 and SEQ ID NO: 6 obtained at a
ratio POSTN/CatK ranging from 250:1 to 5:1 were measured with
LC-MS/MS. Absence of an incomplete digested POSTN fragment (ID SEQ
NO.: 6) served as an indication of complete POSTN digestion at
POSTN/CatK ratio of 5:1 (FIG. 3B).
This data show that the optimal in vitro digestion of human POSTN
appears at POSTN/CatK ratio within a range of 100:1 to 22:1. The
most abundant CatK-digested POSTN fragments are of SEQ ID NO: 2 and
SEQ ID NO: 6.
Example 3
Time Course of CatK-Dependent POSTN Digestion
Time course of appearance of 17 CatK-dependent POSTN fragments
obtained at POSTN/CatK ratio of 50:1, separated and identified with
the use of LC-MS/MS, was evaluated.
CatK digestion, targeted LC-MS/MS and silver stained SDS-PAGE as
described in Example 1. The appearance of 17 different POSTN
fragments were monitored during CatK-dependent POSTN digestion
between 15 min to 2 h, at POSTN/CatK ratio of 50:1 (FIG. 4). The
earliest fragment is of SEQ ID NO: 2 appearing within 15 min from
reaction start, followed by fragments of SEQ ID NO: 6, SEQ ID NO:
3, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 5. Most of the POSTN
digestion finishes within 60 min from the start of reaction.
Those 17 POSTN fragments were then ranked based on their abundance
as measured in LC-MS/MS analysis, as well as speed of their
generation (FIG. 5). Sequences of observed POSTN fragments suggest
that amino acids K, T, E, R and L are preferred cleavage sites for
CatK-digested of POSTN in order of preference as follows
K>T>E>R>L.
The most abundant and fastest appearing CatK-digested POSTN
fragments are of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID
NO: 4, and SEQ ID NO: 5. Therefore, these are preferred POSTN
fragments that could be useful to mark activity of CatK in
bone.
Example 4
Production of Anti-POSTN Fragments Antibodies
Antibodies are generated by conventional immunization protocols.
Specifically, peptides of the invention are coupled to a carrier
molecule, including but not limited to, keyhole limpet hemocianin
and bovine serum albumin (BSA). Peptide fragments, once coupled are
then injected into rabbits for 55 days. The serum of the animals is
collected and purified by immunoaffinity methods against the POSTN
fragments.
Immunization. Two rabbits (H8308 and H8325) were immunized by
injection of the synthetic peptide comprising amidated peptide of
SEQ ID NO: 2, cysteine (C; to allow a conjugation at the N-terminal
end) and 2-aminohexanoic acid (Ahx, hydrophobic spacer allowing
better exposure of the specific sequence), and referred to as
C-Ahx-GSLQPIIK-NH.sub.2, which was further coupled with keyhole
limpet hemocyanin (KLH).
Antibody purification and characterization. The antibodies were
isolated from serum of the animals by immunoaffinity using a gel
coupled with the sequence of peptide 1. Titer of immunopurified
antiserum was evaluated by direct binding on peptide of SEQ ID NO:
2 coupled to bovine serum albumin (BSA), referred to as
BSA-GSLQPIIK. The titer of obtained antiserum was evaluated to be
at about 10.sup.6, supporting that a polyclonal antibody specific
to CatK-digested POSTN fragment of SEQ ID NO: 2 was isolated. Due
to the high sequence homology between human POSTN and POSTN of
other species, those antibodies may react with mouse, rat,
cynomolgous monkey, dog and cat POSTN fragments.
Example 5
CatK-Digested POSTN Fragment Presence in Cortical Mouse Bone
The presence of CatK-digested POSTN fragments of SEQ ID NO: 2 were
assessed in mouse bone using the antibodies prepared according to
Example 4.
Immunohistochemical analysis. The right and left tibiae were
excised from mouse and subsequently fixed in 4% paraformaldehyde
overnight at 4.degree. C. They were then decalcified in 19%
ethylenediaminetetraacetic acid (EDTA) and 4% phosphate-buffered
formalin for 3 weeks. The tibiae were then dehydrated in an
ascending series of ethanol, cleared in Propar (Anatech LTD, Battle
Creek, Mich.), and embedded in paraffin blocks. 8 .mu.m-thick
sections were cut from the blocks at the tibia mid-shaft level
using a RM2155 microtome (Leica, Germany) and mounted on Superfrost
Plus slides (Fisher Scientific, Pittsburg, Pa.). Sections were
air-dried overnight at room temperature. Prior to staining,
sections were incubated in an oven (Hybaib, MGW Biotech) at
60.degree. C. for 1 h, deparaffinized in xylene, and rehydrated in
a descending series of ethanol. De-paraffinized slides were
pre-treated in 3% hydrogen peroxide in methanol to quench
endogeneous peroxidase and rinsed in tap water followed by
non-specific avidin/biotin blocking (Vector Laboratories,
Burlingame, Calif.) according to the manufacturer's directions. All
incubations took place in a humidified chamber. Additional protein
blocking was accomplished with Protein Block-Serum Free (DAKO,
Carpinteria, Calif.). Using the Vectastain Elite ABC (Rabbit IgG)
Kit (Vector Laboratories, Burlingame, Calif.), the slides were
incubated in 1.5% normal goat serum for 30 minutes at room
temperature. The primary antibody (antibody to CatK-digested POSTN
fragment of SEQ ID NO: 2 as obtained in Example 4 was diluted in
Antibody Diluent (DAKO, Carpinteria, Calif.) to a final
concentration of 1/200000 and 1/10000 and incubated at 4.degree. C.
overnight. The following day, slides were rinsed in Wash Buffer
(DAKO, Carpinteria, Calif.) for 15 min on a rocker at room
temperature and incubated in biotinylated goat anti-rabbit
(Vectastain Kit) secondary antibody diluted 1:1000 for 30 min at
room temperature, followed by another rinse in Wash Buffer for 15
min on a rocker at room temperature. The ABC reagent from the
Vector Kit was prepared according to the manufacturer's directions
at a dilution of 1:250 and the slides were incubated in it for 30
min at room temperature and rinsed, as above, in DAKO.RTM. Wash
Buffer. All incubation steps were performed at room temperature and
all rinse steps employed the DAKO.RTM. Wash Buffer at room
temperature on a rocker. Next, slides were incubated in
streptavidin- horseradish peroxidase (HRP) diluted at 1:100 for 30
min, and washed for 15 min. Slides were developed in a working
solution of 3,3'-diaminobenzidine (DAB Substrate Kit for Peroxidase
Kit, Vector Laboratories, Burlingame, Calif.) prepared according to
manufacturer's directions for 10 min at room temperature. Following
a final rinse in deionized water, the slides were mounted in
Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, Mich.). For the
negative control, primary antibody incubation have been replace by
TRIS 0.1M. Digital images were obtained using a microscope with a
camera AxioCam MRc5 controlled by Axiovision AC software (Carl
Zeiss MicroImaging GmbH, Germany).
The presence of CatK-digested POSTN fragments of SEQ ID NO: 2 was
detected at mouse periosteum surface (Ps) of cortical bone region,
specifically in osteocytes and lacuno canalicular system as well as
in the bone matrix with the use of anti-SEQ ID NO: 2 antibodies
used at the concentration of 1/10000, but not at the concentration
of 1/200000 (FIG. 6).
This analysis confirmed that the antibodies of the invention are
able to detect the presence of CatK-digested POSTN fragments of SEQ
ID NO: 2 in mouse osteocytes and lacuno canalicular system of
cortical bone region and that these fragments are specific for this
bone region.
Example 6
ELISA Assay for a CatK-Digested POSTN Fragment
An ELISA assay was developed for assaying CatK-digested POSTN
peptidic fragments of the invention. First, biotinylated peptides
are prepared by conjugating a peptide of the invention to be
assayed with biotin. Those biotinylated POSTN peptides (e.g. 100
.mu.l) are incubated on streptavidin coated microtiter plates (for
2 h, at room temperature, in coating buffer) to coat the plates
with a peptide. After washing off the excess of POSTN peptide used
as calibrator (3-5 times with BSA-Tween blocking solution in TBS
buffer), the sample to be tested (standard solution of the
unmodified POSTN peptide to be assayed, or a buffer sample used as
control solution or a serum sample) is added to each well. Primary
anti-POSTN peptide antibodies prepared as described in Example 4
and specific for the POSTN peptide fragment to be assayed are also
added. For example, 50 .mu.l of standard (synthetic POSTN peptide
to be assayed) at different concentrations of 0, 1, 5, 10, 50, 100,
500, 1000 ng/ml or serum sample and 50 .mu.l of primary H8308/H8325
antibody in assay buffer is added and incubated overnight at
4.degree. C. After incubation, the plates are washed (e.g. 3-5
times with BSA-0.05% Tween blocking solution in TBS buffer) and a
solution of peroxidase-conjugated goat anti-rabbit antibody
(secondary antibody) is pipetted into each well (e.g. 100 .mu.l of
horseradish peroxidase-conjugated goat anti-rabbit antibody diluted
at 1/8000 in TRIS-BSA buffer and incubated for 1 h at room
temperature). After incubation and washing (e.g. 3-5 times with
BSA-0.05% Tween blocking solution in TBS buffer),
H.sub.2O.sub.2/tetramethylbenzidine (TMB) substrate indicator
solution is added (e.g. 100 .mu.l of TMB substrate for 30 min) to
reveal the bound secondary antibody. After incubation at room
temperature for 30 minutes, the colorimetric reaction is stopped by
the addition of 100 .mu.l 2M H.sub.2SO.sub.4, and the optical
density at 405 nm corrected for the absorbance at 650 nm is be
measured.
In such competitive ELISA assay, the peptide POSTN fragment
contained in the sample (free synthetic peptide used as a standard
or the natural peptide fragment comprised in a serum sample)
competes with the biotinylated synthetic peptide coated on the
microtiter plate for the binding to the primary antibody between.
After incubation of a test sample with primary antibodies, the
excess of unbound primary antibodies is removed by washing and the
amount of a primary antibody remaining on the plate is revealed by
the secondary antibody after a colorimetric reaction. The optical
density signal in this case is inversely proportional to the
concentration of the peptide present in the sample.
The specificity of the antibody used in the ELISA is tested by
competition experiments with POSTN fragments generated by
recombinant cathepsin K, synthetic peptides of the invention,
intact recombinant POSTN and the peptides of the invention wherein
one or two amino acids at the C-terminal end are inserted or
deleted. This information is used to control that the assay
recognizes only the identified cathepsin K cleavage site on
POSTN.
Several concentrations of biotinylated peptide, primary and
secondary antibodies are used to optimize the signal to noise
ratio. Different buffers and incubation conditions (time,
temperature) are used to optimize the signal to noise ratio.
Human serum testing is performed to test the peptides of the
invention of sequences selected from group of: SEQ ID NO: 2, SEQ ID
NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. To ensure that the ELISA
provides accurate and reproducible data, the following controls are
performed: 1. Intra and inter assay variability on 3 different
pools of serum samples with different concentrations of endogenous
peptides of the invention. 2. Linearity using serial dilutions of 3
pools of serum samples. 3. Recovery of various concentrations of
synthetic peptides of the invention spiked into 3 pools of human
serum. 4. Stability of serum samples at room temperature, 4.degree.
C. and after 1 to 4 freeze-thaw cycles.
Example 7
ELISA Assay for a CatK-Digested POSTN Fragment of SEQ ID NO: 2
Detection of presence of peptide 1 which was identified as the main
product of the final degradation of human recombinant POSTN by
human Cat-K (Examples 2 and 3) is of interest. Therefore, an
immunoassay ELISA for the quantitative determination of the
presence of peptide 1 in human blood samples was developed as
described in Example 6 and as detailed below.
Sample preparation. Venous human blood samples were collected by
using standardized blood collection tubes for serum (BD VACUTAINER
SST2, advance ref: 367955). Blood was collected between 8 to 10 AM.
Subjects were fasting prior to blood collection for at least 8 h or
overnight. Shortly after, serum separation by centrifugation is
carried out, e.g. 10 min at 3000.times.g, preferably at 4.degree.
C. (2-8.degree. C.) and the obtained serum samples are measured
right after. For longer storage aliquot samples were stored at
-80.degree. C. in 2 ml micro tube with polypropylene cap
(Sarstedt). Samples were not freeze-thaw more than 2 times. Lipemic
or hemolysed samples may give erroneous results and were not used.
Serums samples were mixed well before assaying (Vortex 10 sec).
ELISA protocol. Microtiter wells were washed 3.times. with 300
.mu.l of diluted wash buffer (0.5% PBS BSA, Gibco, cell
signalling). After final wash, remaining wash buffer was removed by
strongly tapping plate against paper towel. Biotinylated synthetic
peptides resulting from the biotinylation of peptide 1 (100 .mu.l
at 2.5 ng/ml in Tris BSA, coating buffer) were incubated on
streptavidin pre-coated microtiter plates (96 wells) for 1 h,
tightly covered, at room temperature (RT) (e.g. 18-26.degree. C.)
with gentle agitation. After incubation, the solution was removed
by aspiration and wells were washed 4.times. with 300 .mu.l diluted
wash buffer, after final wash, remaining wash buffer was removed by
strongly tapping plate against paper towel. 50 .mu.l of standard
(STD) solution of peptide 1 (see below) or control solution (CTRL)
or sample (e.g. serum) was added to the wells. Next, 50 .mu.l of a
polyclonal primary antibody to CatK-digested POSTN fragment of SEQ
ID NO: 2 (obtained in Example 4) diluted to 1/25000 in assay buffer
(TRIS 10 mM, CaCl.sub.2 10 mM, 0.5% BSA, respectively Sigma, MERCK,
Bioconcept Cell Signaling Technology) was added, and the plates
were covered and incubated for 19-20 h at 4.degree. C. (2-8.degree.
C.) under gentle agitation.
Standard (STD) stock solution of peptide 1 at 9.3 mg/ml was diluted
with assay buffer to obtain the following individual standard
solutions STD 5, 15, 30, 60, 120, 240, 480, 960 ng/ml, STD 0
referred to assay buffer. After incubation, the solution was
removed and wells were washed 5.times. with 300 .mu.l of diluted
wash buffer, after final wash remaining wash buffer was removed by
strongly tapping plate against paper towel. Next, 100 .mu.l of a
secondary anti-rabbit antibody conjugated with horseradish
peroxidase (HRP) (peroxydase conjugated affinipure goat anti-rabbit
IGG (H+L), Jacksonlab) at concentration 1/150000 was added to the
well, the plates were covered tightly and incubated for 1 h at RT
under gentle agitation. After incubation, the solution was removed
by aspiration and wells were washed 4.times. with 300 .mu.l diluted
wash buffer. 100 .mu.l of a TMB
(H.sub.2O.sub.2/tetramethylbenzidine) solution (chromogenic
substrate for HRP, Sigma T040) was then added to the wells and
incubated in a dark room between 20-30 min (e.g. 25 min) at RT
under gentle agitation until the colorimetric reaction was stopped
by adding 50 .mu.l of stop solution (H.sub.2SO.sub.4) at
concentration 0.2M. The optical density (OD) of all wells on a
plate reader was measured immediately after the completion of
reactions at 450 nm wavelength. The curve from the OD values of the
STD was constructed and the sample concentration was then deduced
from the so-obtained standard curve (FIG. 7). The assay was
evaluated with 4PL algorithm with various curve fitting
methods.
Analytical Sensitivity
The concentration corresponding to OD (STD 0)-3SD (standard
deviation) of standard 0 as calculated from 15 repeated
measurements of STD 0 was used.
ELISA assay's specificity was analyzed by carrying out the protocol
described above using a sample solution further containing peptide
1 or a control peptide 1 of SEQ ID NO: 20 (modified peptide 1
bearing a C-term, threonine), or another control peptide 2 of SEQ
ID NO: 21 (fragment of peptide 1 missing the C-term Lysine), or
intact POSTN (SEQ ID NO: 1) at concentrations of 60 and 120 ng/ml.
The sample with buffer alone was used as a control and OD values
were measured as a function of the concentration in target peptide
of SEQ ID NO: 2. In another second set of experiments, ELISA assay
was performed on a sample with intact recombinant human POSTN, or
on a sample wherein recombinant human POSTN was digested by
recombinant CatK for 4 hours at a molar ratio of 1/10. A buffer
used for digestion reaction was used as a negative control.
Analytical Reproducibility
Intra-assay variability was assessed on 4 serum samples of
different peptide concentrations run 10-12 times in the same assay.
Inter-assay variability was assessed on 3 serum samples were run
4-10 times in different assays. The coefficient of variability (CV)
was calculated by dividing the SD of the measurements with the mean
value of each sample. In general, intra-assay and inter assay % CV
of less than 15 are acceptable.
Assay Linearity
2 serum samples with high concentrations of endogenous peptide 1
were serially diluted in the assay buffer. The mean % recovery was
then calculated by dividing the measured concentration by the
expected value taking into account the dilution factor. Recoveries
ranging from 80 to 120% are considered acceptable for such ELISA
technology.
The obtained standard curve for peptide 1 is consistent with
standard curves provided by final release quality control (QC)
protocols (OD values of 1.40 or higher obtained for the zero
standard). The analytical lower limit of detection (LLOD) for this
ELISA assay was estimated to be 9.1 ng/ml. It was estimated using
the concentration of the target peptide corresponding to the OD
value of the 0 standard-3 SD. The Lower Limit of Quantification
(LLOQ) was estimated to be 12 ng/ml. The LLOQ is the concentration
of the target peptide in serum samples that can be measured with a
% CV below 20%.
The specificity of this ELISA assay was supported since it did not
detect control peptide SEQ ID NO: 20, control peptide of SEQ ID NO:
21 nor intact POSTN of SEQ ID NO: 1 at the tested concentrations
and was only sensitive for peptide 1 (FIG. 8). In the second set of
experiments, intact recombinant human POSTN (concentration of 0.04
.mu.g/.mu.l) was not detected by ELISA but in contrast, in a sample
where intact POSTN was digested by CatK, CatK-digested POSTN
fragment of SEQ ID NO: 2 was detected at concentration of 29 ng/ml.
The intra-assay CV was below 12.5% and the inter-assay CV for serum
sample above the LLOQ (i.e. 12 ng/ml) was below 14 (Table 2).
TABLE-US-00002 TABLE 2 Mean concentration (ng/ml) N CV %
intra-assay 32.7 10 8.0 50.8 10 9.1 132.9 10 12.4 201 12 11.7
inter-assay 7.5 8 17.1 27.2 10 13.8 42.8 4 10.6
TABLE-US-00003 TABLE 3 Peptide of Serum SEQ ID NO: 2 % ID Dilution
(ng/ml) recovery I Neat (high natural concentration) 201 1/2 104
103 1/4 55.1 110 1/8 31.1 124 II Neat (high natural concentration)
314 1/2 187 119 1/4 84 107 1/8 39 99
Those data support that competitive ELISA assay according to the
invention can reliably and specifically detect peptide 1 in human
serum samples.
Example 8
Presence of CatK-Digested POSTN Fragment of SEQ ID NO: 2 in a
Population of Retired Subjects
The presence of peptide 1 was measured with an ELISA assay of the
invention as described in Example 7. The serum of 195 randomly
chosen subjects of the Geneva Retiree Cohort (GERICO) comprising
more than 1'000 subjects (3/4 females) from the general population
with a mean age of 65.0.+-.1.40 years for 160 women and 35 men was
collected and assayed.
Statistical Analysis
Statistical analyses were performed using MedCalc Statistical
Software version 13.1.2 (MedCalc Software bvba, Ostend, Belgium).
All data were reported as means.+-.SD. Normal distribution was
evaluated by d'Agostino-Pearson test. To take into account that not
all variables were normally distributed, the differences were
assessed by a Mann-Whitney U test. A one way ANOVA with the
tertiles of periostin fragment serum levels (peptide 1) employed as
a factor was performed. Correlations of bone microstructure and
peptide 1 serum levels with bone parameters were analyzed by single
and multivariate linear regression analyses. P<0.05 was
considered the level of statistical significance for regression
coefficient (or .beta. values).
The measured mean concentration of the peptide 1 in those subjects'
serum was at 38.8 ng/ml with respectively the lowest and highest
value of 10.9 and 170.3 ng/ml as shown in Table 4 below. Peptide
concentration value did significantly differ between men and women
and is not correlated to age. Table 5 shows the distribution of
peptide 1 concentrations among the subject's population.
TABLE-US-00004 TABLE 4 Sample size 195 Lowest value 10.93 Highest
value 170.29 Arithmetic mean 38.7869 95% CI for the mean 35.9592 to
41.6147 Median 35.1500 95% CI for the median 32.3065 to 37.0204
Variance 400.8466 Standard deviation 20.0212 Relative standard
deviation 0.5162 (51.62%) Standard error of the mean 1.4337
Coefficient of Skewness 2.4346 (P < 0.0001) Coefficient of
Kurtosis 11.0284 (P < 0.0001) D'Agostino-Pearson test reject
Normality (P < 0.0001) for Normal distribution
TABLE-US-00005 TABLE 5 Percentiles Peptide 1 (ng/ml) 95% CI 2.5
13.0462 11.5298 to 14.4720 5 14.8700 13.0200 to 19.4620 10 20.2800
16.7141 to 22.8331 25 25.7150 24.5058 to 27.7527 75 47.6775 41.9709
to 52.2508 90 61.4100 56.5904 to 65.0074 95 67.8150 63.5783 to
86.5678 97.5 85.0588 68.0309 to 143.5525
The amount of the peptide in tested serum was not associated with
total POSTN (FIG. 9) nor with the POSTN measured by POSTN
antibodies from USCN or Biomedica (FIG. 12).
Those data support that an ELISA assay of the invention is capable
of detecting CatK-digested POSTN fragment of SEQ ID NO: 2 in
general population sera with sensitivity to gender population which
is indicative of a potential sensitivity to the bone quality
status.
Example 9
CatK-Digested POSTN Fragment of SEQ ID NO: 2 as Specific Marker of
Cortical Bone Compartment
Various bone markers and bone structure characteristics were
measured in the patients as selected in patients' population of
Example 8 as follows:
CTX (cross-linked C-terminal telopeptide of type I collagen) and
P1NP (procollagen type 1 N-terminal propeptide) are used as markers
of the bone resorption and of the bone formation, respectively were
measured on a Cobas-6000 instrument using Elecsys reagents (Roche
diagnostics). Serum POSTN (sPOSTN) level was measured by ELISA
assay (SEH339Hu, USCN, China).
Bone mineral density (BMD) of the femoral neck was determined by
dual-energy x-ray absorptiometry (DXA) using a Hologic QDR
Discovery instrument. High-resolution peripheral quantitative
computed tomography measurements were performed using an XtremeCT
instrument (Scanco Medical AG, Basserdorf Switzerland). Scanning of
the immobilized non-dominant forearm and distal tibia was performed
as previously described (Durosier et al., 2013, J Clin Endocrinol
Metab, 98(9): 3873-83). Shortly, a stack of 110 computed tomography
(CT) slices was acquired over a 9-mm length with an isotropic voxel
size of 82 mm, starting proximally at 9.5 and 22.5 mm from a joint
margin reference line for distal radius and distal tibia,
respectively. Reproducibility assessed with repositioning was
0.6-1.0% and 2.8-4.9% for density variables and trabecular (Tb.)
and cortical (Ct.) microstructures, respectively. Tb. and Ct.
microstructure parameters (Bone volume on total volume (BV/TV), Tb.
or Ct. area (Tb./Ct.Ar), Tb. or Ct. thickness (Ct.Th), Moment of
Inertia (MI) and porosity were evaluated as previously described
(Chevalley et al., 2013, Bone, 55(2): 377-83).
Cross sectional (Tables 6) analyses were carried out to determine
whether this peptide could be an independent and specific marker of
cortical bone compartment.
Correlations of peptide 1 and sPOSTN with bone parameters were
analyzed as described in Example 8. All data were reported as
means+/-SD. To take into account that not all variables were
normally distributed, the differences were assessed by a
d'Agostino-Pearson test. CTX adjustment of the measured levels of
peptide 1 was performed by covariance in order to demonstrate that
the association of periostin with bone structure is independent of
classical bone turnover markers such as CTX.
TABLE-US-00006 TABLE 6 peptide 1 peptide 1*/ peptide 1 sPOSTN
sPOSTN peptide 1* sPOSTN* sPOSTN* Age (years) 0.09 0.002 0.06 0.09
-0.005 0.05 (p = 0.48) (p = 0.19) (p = 0.96) (p = 0.44) (p = 0.21)
(p = 0.93) CTX (ng/l) -0.09 -0.08 -0.06 -- -- -- (p = 0.20) (p =
0.17) (p = 0.39) P1NP (.mu.g/l) -0.04 -0.03 -0.03 0.07 0.05 0.04 (p
= 0.55) (p = 0.57) (p = 0.65) (p = 0.35) (p = 0.41) (p = 0.60)
Peptide 1 -- -0.01 0.77 -- -0.01 0.76 (ng/ml) (p = 0.81) (p <
0.0001) (p = 0.87) (p < 0.0001) sPOSTN -0.01 -- -0.50 -0.02 --
-0.50 (ng/ml) (p = 0.84) (p < 0.0001) (p = 0.80) (p < 0.0001)
BMD neck 0.06 0.09 -0.05 0.04 0.08 -0.07 (g/cm.sup.2) (p = 0.41) (p
= 0.15) (p = 0.50) (p = 0.60) (p = 0.21) (p = 0.35) BV/TV tibia
-0.008 0.10 -0.11 -0.03 0.10 -0.13 (p = 0.91) (p = 0.11) (p = 0.13)
(p = 0.70) (p = 0.11) (p = 0.08) Tb. vBMD -0.008 0.12 -0.11 -0.34
0.10 -0.13 (mgHA/cm.sup.3) (p = 0.91) (p = 0.06) (p = 0.13) (p =
0.70) (p = 0.11) (p = 0.08) Ct. area tibia -0.14 0.26 -0.24 -0.18
0.25 -0.28 (mm.sup.2) (p = 0.05) (p < 0.001) (p = 0.0008) (p =
0.01) (p > 0.0001) (p = 0.0002) Ct. vBMD -0.08 0.16 -0.14 -0.14
0.14 -0.17 (mgHA/cm.sup.3) (p = 0.12) (p = 0.01) (p = 0.05) (p =
0.05) (p = 0.03) (p = 0.02) Ct. Porosity -0.07 -0.02 -0.03 -0.05
-0.005 -0.01 (%) tibia (p = 0.36) (p = 0.70) (p = 0.70) (p = 0.51)
(p = 0.93) (p = 0.83) Total polar -0.11 0.27 -0.24 -0.14 0.26 -0.27
MI (mm.sup.4) (p = 0.11) (p < 0.001) (p = 0.0008) (p = 0.05) (p
= 0.001) (p = 0.0003) Failure load -0.07 0.24 -0.21 -0.10 0.23
-0.23 tibia (N) (p = 0.33) (p < 0.01) (p = 0.004) (p = 0.17) (p
= 0.0003) (p = 0.001) Stiffness -0.07 0.24 -0.20 -0.10 0.23 -0.23
tibia (p = 0.35) (p < 0.0001) (p = 0.005) (p = 0.18) (p =
0.0004) (p = 0.001) *CTX adjustment
sPOSTN was positively correlated with cortical area, Ct. vBMD and
fine element analysis parameters (total polar MI, failure load and
stiffness) and not associated with BMD or bone turnover markers
(CTX, P1NP) (Tables 6) which are known markers for bone parameters
but not specific for the cortical bone. Peptide 1 was negatively
associated with cortical parameters (Ct. area tibia, Ct. vBMD,
total polar MI) and not trabecular parameters (Tb. vBMD) confirming
the association of periostin with the cortical compartment. Peptide
1 to sPOSTN ratio, i.e. an index of the digested periostin was
negatively correlated with cortical area, Ct. vBMD, M, failure load
and stiffness and total polar MI. These negative associations
remain significant after adjustment by CTX, indicating that peptide
1 measurement gives additional information than bone remodeling
markers (Table 6). Further, subjects on the high tertile of sPOSTN
have high cortical area and stiffness whereas high tertile Peptide
1 to sPOSTN ratio have significantly lower cortical area and
stiffness (FIG. 10).
Together, the above data indicate that CatK-digested POSTN fragment
of SEQ ID NO: 2 is a specific marker of cortical bone compartment
that provides additional information to known biochemical markers
of bone turnover (CTX, P1NP).
Example 10
CatK-Digested POSTN Fragment of SEQ ID NO: 2 as Specific Marker of
Cortical Bone Compartment (Prospective Analysis)
A prospective analysis of bone markers and parameters as well as
serum levels of peptide 1 and POSTN was carried out on 137 randomly
chosen subjects of the GERICO population as described above. In
this study, bone microstructure measurements (DXA, HRpQCT) were
performed 3 years after the evaluation of bone markers in order to
determine if bone markers are predictive of bone structure. BMD and
tibial bone microarchitecture regression are reported in Table 7 as
monitored with Peptide 1, sPostn and ratio Peptide 1 to sPostn.
TABLE-US-00007 TABLE 7 Peptide 1/ Peptide 1 sPOSTN sPOSTN Age
(years) 0.11 (p = 0.18) 0.03 (p = 0.70) 0.02 (p = 0.84) BMD neck
0.03 (p = 0.70) 0.10 (p = 0.19) -0.09 (p = 0.25) (g/cm.sup.2) BV/TV
tibia -0.04 (p = 0.61) 0.16 (p = 0.03) -0.12 (p = 0.15) BV/TV
radius -0.07 (p = 0.41) 0.20 (p = 0.01) -0.11 (p = 0.19) Ct. area
tibia -0.14 (p = 0.10) 0.30 (p < 0.001) -0.23 (p < 0.01)
(mm.sup.2) Ct. area radius -0.11 (p = 0.23) 0.28 (p < 0.001)
-0.24 (p = 0.006) (mm.sup.2) Ct.Th tibia -0.14 (p = 0.10) 0.21 (p
< 0.01) -0.20 (p = 0.02) (mm.sup.2) Ct.Th radius -0.11 (p =
0.21) 0.20 (p = 0.01) -0.16 (p = 0.06) (mm.sup.2)
sPOSTN was positively correlated BV/TV, Ct. area and CtTh of tibia
and radius and not associated with BMD (Table 7). Peptide 1 was
negatively associated with cortical parameters (Ct. area, CtTh).
Peptide 1 to sPOSTN ratio was negatively correlated with Ct. area
and CtTh of tibia and radius confirming that CatK-digested POSTN
fragment of SEQ ID NO: 2 is a specific marker of cortical bone. The
same analysis is then performed 6 years after the initial
evaluation of bone markers on the cohort of 1'000 subjects.
Example 11
Association of CatK-Digested POSTN Fragment of SEQ ID NO: 2 with
Cortical Bone Compartment are Maintained After Adjustment for POSTN
(Prospective Analysis)
Bone markers and bone structure parameters are analyzed together
with serum levels of peptide 1 and POSTN measured with commercially
available kits (with two commercially available kits: ELISA Kit for
Periostin, Uscn Life Science, Inc. (Product No.: SEH339Hu) (Kit 1),
and ELISA Kit for Periostin, Biomedica Immunoassays (Cat. No.:
BI-20433) (Kit 2) on 165 randomly chosen subjects of the GERICO
population as described above. The measured levels of peptide 1
were adjusted by multiple regression analysis with POSTN level as
detected with kit 1 or kit 2. BMD and bone microarchitecture
regression with peptide 1 and peptide 1 adjusted (adj.) to POSTN
levels measured by kit 1 or 2 are reported in Table 8.
TABLE-US-00008 TABLE 8 peptide 1 - peptide 1 - adj. POSTN adj.
POSTN peptide 1 (kit 1) (kit 2) Age (years) 0.06 p = 0.47 -0.01 p =
0.90 0.06 p = 0.47 BMD neck -0.02 p = 0.78 -0.09 p = 0.26 -0.01 p =
0.87 (g/cm.sup.2) BV/TV tibia -0.07 p = 0.35 -0.12 p = 0.11 -0.07 p
= 0.39 BV/TV radius -0.06 p = 0.44 -0.07 p = 0.36 -0.07 p = 0.41
Ct. area tibia -0.17 p = 0.02 -0.19 p = 0.02 -0.16 p = 0.04
(mm.sup.2) Ct. area radius -0.14 p = 0.10 -0.16 p = 0.05 -0.11 p =
0.16 (mm.sup.2) Ct.Th tibia (mm.sup.2) -0.16 p = 0.03 -0.15 p =
0.05 -0.15 p = 0.09 Ct.Th radius -0.16 p = 0.05 -0.16 p = 0.05
-0.15 p = 0.11 (mm.sup.2)
Peptide 1 (SEQ ID NO: 2) was negatively associated with cortical
parameters (Ct. area tibia, CtTh tibia/radius) and at least for Ct.
area tibia this association remained negative after adjustment of
Peptide 1 level to the POSTN level measured by two different kits.
For CtTh tibia/radius negative association for Peptide 1 remained
after adjustment of Peptide 1 level to the POSTN level measured by
kit 1. Also, after adjustment of Peptide 1 level to the POSTN level
measured by kit 1 negative association of Peptide 1 for Ct. radius
become statistically significant.
The amount of peptide 1 in tested sera was not associated with
total POSTN as measured by kit 1 or 2 (FIG. 11).
This result confirms that developed antibodies used for the
detection of CatK-digested POSTN fragment of the invention in a
serum do not detect the same molecules as the commercial antibodies
and thus are specific. Further, CatK-digested POSTN fragment of the
invention can serve as a specific marker of cortical bone
compartment.
Example 12
Association of CatK-Digested POSTN Fragment of SEQ ID NO: 2 with
Incident Fracture (Prospective Analysis)
An analysis of bone markers and parameters as well as serum levels
of peptide 1 and POSTN were carried out on subjects with incident
fracture and matched controls.
In this study, within GERICO cohort of 759 post-menopausal women
that were followed up to 6 years, 54 incident fracture with low and
middle trauma (osteoporotic fracture; "fracture" group) were
registered (excluding vertebral, toe, finger and skull fractures)
and compared to matched for age and body mass index (BMI) randomly
selected subjects from GERICO (n=198, "non-fracture" group). An
analysis of bone markers and parameters as well as serum levels of
peptide 1 and POSTN was carried out as described in Example 9.
POSTN was measured with ELISA Kit for Periostin, Uscn Life Science,
Inc. (Product No.: SEH339Hu). In order to quantify how strongly
Peptide 1 levels were associated with the presence or absence of
fracture the Odds ratio based on 1 SD of peptide 1 was calculated.
Cox model proportional hazard regression and area under the curve
(AUC) of Receiver Operating Characteristic (ROC) curve was
performed as statistic survival model using MedCalc Statistical
Software version 13.1.2. Survival models relate the time that
passes before some event occurs, here the risk of fracture, to one
or more covariates that may be associated with that quantity of
time.
The fracture risk assessment tool (FRAX) developed by the World
Health Organization (see Worldwide Website: shef.ac.uk/FRAX/) and
country-specific data (Lippuner et al., 2010, Osteoporosis Int. 21:
381-9) was used to assess subjects' risk of fracture using clinical
risk factors such as age, BMI and history of fracture with
inclusion of femoral neck BMD.
Characteristic of the cohort is reported in Table 9 and shows that
the fracture group had lower values of BMD, Tb. and Ct.
microstructure parameters at the tibia and radius (BV/TV, Ct. area,
Ct.Th). Peptide 1 level was significantly higher in the fracture
group versus control group (+53%, p<0.001), whereas the
classical bone turnover markers (CTX, P1NP) and the total POSTN
were not significantly different between the two groups.
TABLE-US-00009 TABLE 9 Fracture Non fracture P value Age (years)
65.2 .+-. 1.4 65.0 .+-. 1.4 0.19 BMI 25.2 .+-. 4.6 25.0 .+-. 4.1
0.81 BMD neck (g/cm.sup.2) 0.6764 .+-. 0.11 0.7245 .+-. 0.11 0.004
BV/TV tibia 0.117 .+-. 0.03 0.127 .+-. 0.03 0.01 BV/TV radius 0.103
.+-. 0.03 0.119 .+-. 0.029 0.001 Ct. area tibia (mm.sup.2) 87.70
.+-. 23.13 106.54 .+-. 28.37 0.001 Ct. area radius (mm.sup.2) 43.82
.+-. 12.19 54.20 .+-. 13.74 <0.001 Ct.Th tibia (mm.sup.2) 0.8442
.+-. 0.25 1.007 .+-. 0.25 <0.001 Ct.Th radius (mm.sup.2) 0.6432
.+-. 0.196 0.7682 .+-. 0.1681 <0.001 Ct. Porosity (%) tibia
0.094 .+-. 0.037 0.082 .+-. 0.03 0.01 Ct. Porosity (%) radius 0.028
.+-. 0.01 0.026 .+-. 0.01 0.35 CTX (ng/L) 383.17 .+-. 202 362.43
.+-. 216 0.51 P1NP (ug/L) 45.94 .+-. 21.14 46.62 .+-. 25.43 0.56
POSTN (ng/ml) 431.88 .+-. 191 412 .+-. 165 0.52 Peptide 1 (ng/ml)
63.30 .+-. 42.37 41.38 .+-. 25.84 <0.001
Cox proportional hazard regression was obtained as reported in
Table 10 and showed a positive association of Peptide 1 and
incident fracture with a hazard ratio of 1.58 [1.24-2.01]
indicating that with an increase of 1 standard deviation of peptide
1, one increased risk of fracture by 58% (Table 10 A). Furthermore,
with the highest tertiles of Peptide 1 the risk of fracture was
further increased with a hazard ratio of 4.74 [2.16-10.40].
Predictions of fracture by Peptide 1 remained significant after
adjustment by BMD neck, Ct. area radius, BV/TV radius or CTX marker
(Table 10 B-E).
TABLE-US-00010 TABLE 10 Hazard ratio P value A. Cox Hazard ratio
(Independent) Peptide 1 (ng/ml) 1.58 [1.24-2.01] 0.001 BMD neck
(g/cm2) 0.72 [0.54-0.96] P < 0.01 CTX (ng/L) 0.97 [0.92-1.03]
0.39 B. Cox Hazard ratio (model) Peptide 1 (ng/ml) 1.62 [1.27-2.07]
P < 0.001 BMD neck (g/cm2) 0.72 [0.54-0.97] P < 0.01 C. Cox
Hazard ratio (model) Peptide 1 (ng/ml) 1.58 [1.23-2.02] P <
0.001 Ct. area radius 0.45 [0.32-0.64] P < 0.001 D. Cox Hazard
ratio (model) Peptide 1 (ng/ml) 1.54 [1.20-1.95] P < 0.001 BV/TV
radius 0.98 [0.98-0.99] P < 0.05 E. Cox Hazard ratio (model)
Peptide 1 (ng/ml) 1.59 [1.24-2.02] P < 0.001 CTX (ng/L) 1.006
[0.95-1.06] P < 0.01
The ROC curve illustrates the true positive rate (Sensitivity) in
function of the false positive rate (1-Specificity) for different
cut-off points and thus allows appreciating how well a parameter
can distinguish between fracture and non-fracture group by
measuring the area under the curve (AUC). Individually, Peptide 1
as well as standard clinical measures such as BMD and FRAX
significantly discriminated fracture events (Table 11). However,
peptide 1 combined with BMD and peptide 1 combined with FRAX
discriminated more subjects with fracture than BMD or FRAX alone
(FIG. 11).
TABLE-US-00011 TABLE 11 ROC Odds ratio AUC P value Peptide 1 1.76
[1.27-2.43] 0.6771 0.001 (ng/ml) BMD neck 0.15 [0.02-1.28] 0.5548 P
= 0.08 (g/cm.sup.2) FRAX 2.23 .times. 10.sup.-7 [2.16 .times.
10.sup.-11-0.0002] 0.6668 0.001
This result confirms that developed antibodies used for the
detection of CatK-digested POSTN fragment of the invention in a
serum can be used as an additional tool to identify patients which
at risk of getting an osteoporotic fracture. This higher
association of CatK-digested POSTN fragment of the invention with
incident fracture as compared to the cortical structure parameters
indicates that CatK-digested POSTN fragment of the invention do not
only serves as a marker of bone structure but also as a marker of
bone quality.
LIST OF SEQUENCES
TABLE-US-00012 Homo sapiens periostin SEQ ID NO: 1
MIPFLPMFSLLLLLIVNPINANNHYDKILAHSRIRGRDQGPNVCALQQI
LGIKKKYFSICKNWYKKSICGQKTIVLYECCPGYMRMEGMKGCPAVLPI
DHVYGILGIVGATTIQRYSDASKLREEIEGKGSFTYFAPSNEAWDNLDS
DIRRGLESNVNVELLNALHSHMINKRMLIKDLKNGMIIPSMYNNLGLFI
NHYPNGVVIVNCARIIHGNQIAINGVVHVIDRVLIQIGTSIQDFIEAED
DLSSFRAAAITSDILEALGRDGHFILFAPTNEAFEKLPRGVLERIMGDK
VASEALMKYHILNTLQCSESIMGGAVFETLEGNTIEIGCDGDSITVNGI
KMVNKKDIVINNGVIHLIDQVLIPDSAKQVIELAGKQQTTFIDLVAQLG
LASALRPDGEYILLAPVNNAFSDDILSMDQRLLKLILQNHILKVKVGLN
ELYNGQILETIGGKQLRVFVYRTAVCIENSCMEKGSKQGRNGAIHIFRE
IIKPAEKSLHEKLKQDKRFSTFLSLLEAADLKELLTQPGDWILFVPIND
AFKGMTSEEKEILIRDKNALQNIILYHLTPGVFIGKGFEPGVINILKTI
QGSKIFLKEVNDILLVNELKSKESDIMTINGVIHVVDKLLYPADTPVGN
DQLLEILNKLIKYIQIKFVRGSTFKEIPVIVYTTKIITKVVEPKIKVIE
GSLQPIIKTEGPTLIKVKIEGEPEFRLIKEGETITEVIHGEPIIKKYTK
IIDGVPVEITEKETREERIITGPEIKYTRISIGGGETEETLKKLLQEEV
IKVIKFIEGGDGHLFEDEEIKRLLQGDTPVRKLQANKKVQGSRRRLREG RSQ Peptide 1
(Periostin fragment 1) SEQ ID NO: 2 GSLQPIIK Peptide 2 (Periostin
fragment 2) SEQ ID NO: 3 EEIEGKGSFT Peptide 3 (Periostin fragment
3) SEQ ID NO: 4 TTQGSKIF Peptide 4 (Periostin fragment 4) SEQ ID
NO: 5 NEAFEKLPR Peptide 5 (Periostin fragment 5) SEQ ID NO: 6
GSLQPIIKTEGPT Peptide 6 (Periostin fragment 6) SEQ ID NO: 7
SEEKEILIR Peptide 7 (Periostin fragment 7) SEQ ID NO: 8 AADLKELL
Peptide 8 (Periostin fragment 8) SEQ ID NO: 9 TGGGETEETLK Peptide 9
(Periostin fragment 9) SEQ ID NO: 10 TTQGSKIFL Peptide 10
(Periostin fragment 10) SEQ ID NO: 11 SALRPDGEYTLL Peptide 11
(Periostin fragment 11) SEQ ID NO: 12 EGETITEVIHGEPIIK Peptide 12
(Periostin fragment 12) SEQ ID NO: 13 YECCPGYMR Peptide 13
(Periostin fragment 13) SEQ ID NO: 14 AADLKELLT Peptide 14
(Periostin fragment 14) SEQ ID NO: 15 IGCDGDSITVNGIK Peptide 15
(Periostin fragment 15) SEQ ID NO: 16 NDAFKGMT Peptide 16
(Periostin fragment 16) SEQ ID NO: 17 AEDDLSSFR Peptide 17
(Periostin fragment 17) SEQ ID NO: 18 DTLLVNELK Homo sapiens CatK
(AAH16058.1) SEQ ID NO: 19
Mwglkvlllpvvsfalypeeildthwelwkkthrkgynnkvdeisrrli
weknlkyisihnleaslgvhtyelamnhlgdmtseevvqkmtglkvpls
hsrsndtlyipewegrapdsvdyrkkgyvtpvknqgqcgscwafssvga
legglkkktgkllnlspqnlvdcvsendgcgggymtnafgyvqknrgid
sedaypyvggeescmynptgkaakorgyreipegnekalkravarvgpv
svaidasltsfqfyskgvyydescnsdnlnhavlavgygiqkgnkhwii
knswgenwgnkgyilmarnknnacgianlasfpkm Control Peptide 1, SEQ ID NO:
20 GSLQPIIKT Control Peptide 2, SEQ ID NO: 21 GSLQPII Peptide 18
(Periostin fragment 18) SEQ ID NO: 22 INGVVHVIDRVLT Peptide 19
(Periostin fragment 19) SEQ ID NO: 23 TSIQDFIEAEDDLSSFR Peptide 20
(Periostin fragment 20) SEQ ID NO: 24 ATTTQRYSDASKLR Peptide 21
(Periostin fragment 21) SEQ ID NO: 25 APTNEAFEKLPR
SEQUENCE LISTINGS
1
251836PRTHomo sapiens 1Met Ile Pro Phe Leu Pro Met Phe Ser Leu Leu
Leu Leu Leu Ile Val1 5 10 15Asn Pro Ile Asn Ala Asn Asn His Tyr Asp
Lys Ile Leu Ala His Ser 20 25 30Arg Ile Arg Gly Arg Asp Gln Gly Pro
Asn Val Cys Ala Leu Gln Gln 35 40 45Ile Leu Gly Thr Lys Lys Lys Tyr
Phe Ser Thr Cys Lys Asn Trp Tyr 50 55 60Lys Lys Ser Ile Cys Gly Gln
Lys Thr Thr Val Leu Tyr Glu Cys Cys65 70 75 80Pro Gly Tyr Met Arg
Met Glu Gly Met Lys Gly Cys Pro Ala Val Leu 85 90 95Pro Ile Asp His
Val Tyr Gly Thr Leu Gly Ile Val Gly Ala Thr Thr 100 105 110Thr Gln
Arg Tyr Ser Asp Ala Ser Lys Leu Arg Glu Glu Ile Glu Gly 115 120
125Lys Gly Ser Phe Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp Asp Asn
130 135 140Leu Asp Ser Asp Ile Arg Arg Gly Leu Glu Ser Asn Val Asn
Val Glu145 150 155 160Leu Leu Asn Ala Leu His Ser His Met Ile Asn
Lys Arg Met Leu Thr 165 170 175Lys Asp Leu Lys Asn Gly Met Ile Ile
Pro Ser Met Tyr Asn Asn Leu 180 185 190Gly Leu Phe Ile Asn His Tyr
Pro Asn Gly Val Val Thr Val Asn Cys 195 200 205Ala Arg Ile Ile His
Gly Asn Gln Ile Ala Thr Asn Gly Val Val His 210 215 220Val Ile Asp
Arg Val Leu Thr Gln Ile Gly Thr Ser Ile Gln Asp Phe225 230 235
240Ile Glu Ala Glu Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala Ile Thr
245 250 255Ser Asp Ile Leu Glu Ala Leu Gly Arg Asp Gly His Phe Thr
Leu Phe 260 265 270Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg
Gly Val Leu Glu 275 280 285Arg Ile Met Gly Asp Lys Val Ala Ser Glu
Ala Leu Met Lys Tyr His 290 295 300Ile Leu Asn Thr Leu Gln Cys Ser
Glu Ser Ile Met Gly Gly Ala Val305 310 315 320Phe Glu Thr Leu Glu
Gly Asn Thr Ile Glu Ile Gly Cys Asp Gly Asp 325 330 335Ser Ile Thr
Val Asn Gly Ile Lys Met Val Asn Lys Lys Asp Ile Val 340 345 350Thr
Asn Asn Gly Val Ile His Leu Ile Asp Gln Val Leu Ile Pro Asp 355 360
365Ser Ala Lys Gln Val Ile Glu Leu Ala Gly Lys Gln Gln Thr Thr Phe
370 375 380Thr Asp Leu Val Ala Gln Leu Gly Leu Ala Ser Ala Leu Arg
Pro Asp385 390 395 400Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn
Ala Phe Ser Asp Asp 405 410 415Thr Leu Ser Met Asp Gln Arg Leu Leu
Lys Leu Ile Leu Gln Asn His 420 425 430Ile Leu Lys Val Lys Val Gly
Leu Asn Glu Leu Tyr Asn Gly Gln Ile 435 440 445Leu Glu Thr Ile Gly
Gly Lys Gln Leu Arg Val Phe Val Tyr Arg Thr 450 455 460Ala Val Cys
Ile Glu Asn Ser Cys Met Glu Lys Gly Ser Lys Gln Gly465 470 475
480Arg Asn Gly Ala Ile His Ile Phe Arg Glu Ile Ile Lys Pro Ala Glu
485 490 495Lys Ser Leu His Glu Lys Leu Lys Gln Asp Lys Arg Phe Ser
Thr Phe 500 505 510Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys Glu Leu
Leu Thr Gln Pro 515 520 525Gly Asp Trp Thr Leu Phe Val Pro Thr Asn
Asp Ala Phe Lys Gly Met 530 535 540Thr Ser Glu Glu Lys Glu Ile Leu
Ile Arg Asp Lys Asn Ala Leu Gln545 550 555 560Asn Ile Ile Leu Tyr
His Leu Thr Pro Gly Val Phe Ile Gly Lys Gly 565 570 575Phe Glu Pro
Gly Val Thr Asn Ile Leu Lys Thr Thr Gln Gly Ser Lys 580 585 590Ile
Phe Leu Lys Glu Val Asn Asp Thr Leu Leu Val Asn Glu Leu Lys 595 600
605Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly Val Ile His Val Val
610 615 620Asp Lys Leu Leu Tyr Pro Ala Asp Thr Pro Val Gly Asn Asp
Gln Leu625 630 635 640Leu Glu Ile Leu Asn Lys Leu Ile Lys Tyr Ile
Gln Ile Lys Phe Val 645 650 655Arg Gly Ser Thr Phe Lys Glu Ile Pro
Val Thr Val Tyr Thr Thr Lys 660 665 670Ile Ile Thr Lys Val Val Glu
Pro Lys Ile Lys Val Ile Glu Gly Ser 675 680 685Leu Gln Pro Ile Ile
Lys Thr Glu Gly Pro Thr Leu Thr Lys Val Lys 690 695 700Ile Glu Gly
Glu Pro Glu Phe Arg Leu Ile Lys Glu Gly Glu Thr Ile705 710 715
720Thr Glu Val Ile His Gly Glu Pro Ile Ile Lys Lys Tyr Thr Lys Ile
725 730 735Ile Asp Gly Val Pro Val Glu Ile Thr Glu Lys Glu Thr Arg
Glu Glu 740 745 750Arg Ile Ile Thr Gly Pro Glu Ile Lys Tyr Thr Arg
Ile Ser Thr Gly 755 760 765Gly Gly Glu Thr Glu Glu Thr Leu Lys Lys
Leu Leu Gln Glu Glu Val 770 775 780Thr Lys Val Thr Lys Phe Ile Glu
Gly Gly Asp Gly His Leu Phe Glu785 790 795 800Asp Glu Glu Ile Lys
Arg Leu Leu Gln Gly Asp Thr Pro Val Arg Lys 805 810 815Leu Gln Ala
Asn Lys Lys Val Gln Gly Ser Arg Arg Arg Leu Arg Glu 820 825 830Gly
Arg Ser Gln 83528PRTArtificial SequencePeriostin fragment 1 2Gly
Ser Leu Gln Pro Ile Ile Lys1 5310PRTArtificial SequencePeriostin
fragment 2 3Glu Glu Ile Glu Gly Lys Gly Ser Phe Thr1 5
1048PRTArtificial SequencePeriostin fragment 3 4Thr Thr Gln Gly Ser
Lys Ile Phe1 559PRTArtificial SequencePeriostin fragment 4 5Asn Glu
Ala Phe Glu Lys Leu Pro Arg1 5613PRTArtificial SequencePeriostin
fragment 5 6Gly Ser Leu Gln Pro Ile Ile Lys Thr Glu Gly Pro Thr1 5
1079PRTArtificial SequencePeriostin fragment 6 7Ser Glu Glu Lys Glu
Ile Leu Ile Arg1 588PRTArtificial SequencePeriostin fragment 7 8Ala
Ala Asp Leu Lys Glu Leu Leu1 5911PRTArtificial SequencePeriostin
fragment 8 9Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu Lys1 5
10109PRTArtificial SequencePeriostin fragment 9 10Thr Thr Gln Gly
Ser Lys Ile Phe Leu1 51112PRTArtificial SequencePeriostin fragment
10 11Ser Ala Leu Arg Pro Asp Gly Glu Tyr Thr Leu Leu1 5
101216PRTArtificial SequencePeriostin fragment 11 12Glu Gly Glu Thr
Ile Thr Glu Val Ile His Gly Glu Pro Ile Ile Lys1 5 10
15139PRTArtificial SequencePeriostin fragment 12 13Tyr Glu Cys Cys
Pro Gly Tyr Met Arg1 5149PRTArtificial SequencePeriostin fragment
13 14Ala Ala Asp Leu Lys Glu Leu Leu Thr1 51514PRTArtificial
SequencePeriostin fragment 14 15Ile Gly Cys Asp Gly Asp Ser Ile Thr
Val Asn Gly Ile Lys1 5 10168PRTArtificial SequencePeriostin
fragment 15 16Asn Asp Ala Phe Lys Gly Met Thr1 5179PRTArtificial
SequencePeriostin fragment 16 17Ala Glu Asp Asp Leu Ser Ser Phe
Arg1 5189PRTArtificial SequencePeriostin fragment 17 18Asp Thr Leu
Leu Val Asn Glu Leu Lys1 519329PRTArtificial SequenceHomo sapiens
CatK 19Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala
Leu1 5 10 15Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys
Lys Thr 20 25 30His Arg Lys Gln Tyr Asn Asn Lys Val Asp Glu Ile Ser
Arg Arg Leu 35 40 45Ile Trp Glu Lys Asn Leu Lys Tyr Ile Ser Ile His
Asn Leu Glu Ala 50 55 60Ser Leu Gly Val His Thr Tyr Glu Leu Ala Met
Asn His Leu Gly Asp65 70 75 80Met Thr Ser Glu Glu Val Val Gln Lys
Met Thr Gly Leu Lys Val Pro 85 90 95Leu Ser His Ser Arg Ser Asn Asp
Thr Leu Tyr Ile Pro Glu Trp Glu 100 105 110Gly Arg Ala Pro Asp Ser
Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr 115 120 125Pro Val Lys Asn
Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser 130 135 140Val Gly
Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu145 150 155
160Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly
165 170 175Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln
Lys Asn 180 185 190Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val
Gly Gln Glu Glu 195 200 205Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala
Ala Lys Cys Arg Gly Tyr 210 215 220Arg Glu Ile Pro Glu Gly Asn Glu
Lys Ala Leu Lys Arg Ala Val Ala225 230 235 240Arg Val Gly Pro Val
Ser Val Ala Ile Asp Ala Ser Leu Thr Ser Phe 245 250 255Gln Phe Tyr
Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp 260 265 270Asn
Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly Ile Gln Lys Gly 275 280
285Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn
290 295 300Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys Asn Asn Ala Cys
Gly Ile305 310 315 320Ala Asn Leu Ala Ser Phe Pro Lys Met
325209PRTArtificial SequenceControl Peptide 1 20Gly Ser Leu Gln Pro
Ile Ile Lys Thr1 5217PRTArtificial SequenceControl Peptide 2 21Gly
Ser Leu Gln Pro Ile Ile1 52213PRTArtificial SequencePeriostin
fragment 18 22Thr Asn Gly Val Val His Val Ile Asp Arg Val Leu Thr1
5 102317PRTArtificial SequencePeriostin fragment 19 23Thr Ser Ile
Gln Asp Phe Ile Glu Ala Glu Asp Asp Leu Ser Ser Phe1 5 10
15Arg2414PRTArtificial SequencePeriostin fragment 20 24Ala Thr Thr
Thr Gln Arg Tyr Ser Asp Ala Ser Lys Leu Arg1 5 102512PRTArtificial
SequencePeriostin fragment 21 25Ala Pro Thr Asn Glu Ala Phe Glu Lys
Leu Pro Arg1 5 10
* * * * *